Novel polyamine analog conjugates and quinone conjugates as therapies for cancers and prostate diseases

ABSTRACT

Peptide conjugates in which cytocidal and cytostatic agents, such as polyamine analogs or naphthoquinones, are conjugated to a polypeptide recognized and cleaved by enzymes such as prostate-specific antigen (PSA) and cathepsin B are provided, as well as compositions comprising these conjugates. Methods of using these conjugates in the treatment of prostate diseases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending U.S. Provisional PatentApplication Ser. No. 60/131,809 filed Apr. 30, 1999. The content of thatapplication is hereby incorporated by reference herein in its entirety.This application also incorporates by reference U.S. Ser. No. 60/131,779(Attorney Docket No. 376463000400) and U.S. Ser. No. 60/131,842(Attorney Docket No. 376463000500), also filed on Apr. 30, 1999. Thecontents of those applications are hereby incorporated by referenceherein in their entirety. This application also incorporates byreference U.S. Ser. No. ______ (Attorney Docket No. 376462000400) andU.S. Ser. No. ______ (Attorney Docket No. 376462000500) co-filed withthis application on Apr. 27, 2000. The contents of those applicationsare also hereby incorporated by reference herein in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable

TECHNICAL FIELD

This invention relates to therapeutic compositions in which a cytostaticor cytocidal compound, such as a polyamine analog or a quinone, isconjugated to a polypeptide recognized and cleaved by enzymes such asprostate specific antigen (PSA) and cathepsin B. This invention alsorelates to medicinal uses of these conjugates, such as uses in treatingcancer, and uses in treating prostate diseases such as prostate cancer,prostatitis and benign prostatic hyperplasia (BPH).

BACKGROUND OF THE INVENTION

Despite advances in early diagnosis, prostate cancer remains a diseasewith high and increasing annual incidence and mortality. Prostate canceris now the most frequently diagnosed cancer in men. This cancer is oftenlatent; many men carry prostate cancer cells without overt signs ofdisease. Autopsies of individuals dying of other causes show prostatecancer cells in 30% of men at age 50; by age 80, the prevalence is 60%.Further, prostate cancer can take up to 10 years to kill the patientafter initial diagnosis. Prostate cancer is newly diagnosed in over180,000 men in the U.S. each year, of which over 39,000 will die of thedisease. In early stage cancers, metastasis occurs to lymph nodes. Inlate stage, metastasis to bone is common and often associated withuncontrollable pain.

In addition to cancer, two other significant diseases of the prostateare BPH and prostatitis. The cost of treating these three diseases isimmense. The annual treatment of prostatic diseases in the U.S. requiresabout 4.4 million physician visits and 850,000 hospitalizations, andcosts billions of dollars. Although treatments for prostatic diseasesexist, these are generally only partially or temporarily effectiveand/or produce unacceptable side effects.

Benign prostatic hyperplasia (BPH) causes urinary obstruction, resultingin urinary incontinence. It occurs in almost 80% of men by the age of80. BPH is often treated surgically with a transurethral resection ofthe prostate (TURP). This procedure is very common: 500,000 TURPs areperformed in the U.S. each year and BPH is the second most common causeof surgery in males. Unfortunately, a side-effect of TURP is theelimination of the ejaculatory ducts and the nerve bundles of the penis,resulting in impotence in 90% of patients.

An alternative therapy for prostate cancer involves radiation therapy. Acatheter has been developed which squeezes prostate tissue duringmicrowave irradiation; this increases the therapeutic temperature towhich the prostate tissue more distal to the microwave antennae can beheated without excessively heating nearby non-prostate tissue. U.S. Pat.No. 5,007,437. A combination of a radiating energy device integratedwith a urinary drainage Foley type catheter has also been developed.U.S. Pat. No. 5,344,435. However, cancerous prostatic cells generallydemonstrate a slow growth rate; few cancer cells are actively dividingat any one time. As a result, prostate cancer is generally resistant toradiation therapy.

This slow growth rate also makes prostate cancer resistant tochemotherapy, although several such methods are now in use or indevelopment. Pharmacotherapy for the treatment of BPH is currently aimedat relaxing prostate smooth muscle (alpha₁ blockade) and decreasingprostate volume (androgen suppression). Clinical trials have beenundertaken to evaluate selective alpha, blockers, antiandrogens, and5-alpha reductase inhibitors for the treatment of BPH. Finasteride, a5-alpha reductase inhibitor, has shown an ability to cause regression ofthe hyperplastic prostate gland in a majority of patients. Mocellini et.al. (1993) Prostate 22:291; and Marberger (1998) Urology 51:677-86.

Additional therapeutic techniques for prostate cancer include usingchemical forms of medical castration by shutting down androgenproduction in the testes, or directly blocking androgen production inthe prostate. For the treatment of prostate cancer oral estrogens andluteinizing releasing hormone analogs are used as well as surgicalremoval of glands that produce androgens (orchiectomy or adrenalectomy).However, estrogens are no longer recommended because of serious, evenlethal, cardiovascular complications. Luteinizing hormone releasinghormone (LHRH) analogs are used instead. However, hormonal therapyinvariably fails with time with the development of hormone-resistanttumor cells. Furthermore, since 20% of patients fail to respond tohormonal therapy, it is believed that hormone-resistant cells arepresent at the onset of therapy.

Estramustine, a steroidal nitrogen mustard derivative, was originallythought to be suitable for targeted drug delivery through conjugation ofestrogen to toxic nitrogen mustard. Clinical trials, however, have beendisappointing when survival is used as an endpoint. Finasteride, a 4-azasteroid (Proscar® from Merck & Co.), inhibits the enzyme responsible forthe intracellular conversion of testosterone to dihydrotestosterone, themost potent androgen in the prostate. Casodex® (bicalutamide, Zeneca,Ltd.), a non-steroidal anti-androgen, is thought to inhibit cellularuptake of testosterone by blocking androgen receptors in the nucleus.However, almost all advanced cancer prostate cells fail to respond toandrogen deprivation.

An additional method for treating prostatic diseases involvesadministration of inhibitors of polyamine synthesis. Dunzendorfer (1985)Urol. Int. 40:241-250. Naturally-produced polyamines include spermidineand spermine and their precursor, diamine putrescine, which are secretedby the prostate gland and are abundant in the seminal fluid. Polyaminesare required for cell division, and probably for differentiation.Spermine apparently stabilizes the DNA, which is tightly packed in theheads of sperm cells. Polyamines may be essential for stability of actinfilament bundles and microtubules. However, polyamine biosynthesisinhibitors such as alpha-difluoromethylornithine (DFMO) causetoxicities, including severe hearing loss, these toxicities sometimesforcing the cessation of treatment. Splinter et al. (1986) Eur. J.Cancer Clin. Oncol. 22:61-67; and Horn et al. (1987) Eur. J. CancerClin. Oncol. 23:1103-1107. Another inhibitor,methylglyoxal-bis-guanylhydrazone (MGBG), caused side effects so extremethat, in one study, drug deaths occurred in over half of treatedanimals. Dunzendorfer (1985); and Herr et al. (1984) Cancer53:1294-1298.

A related type of therapy for prostate cancer involves using polyamineanalogs, such as DENSPM (N1,N11-diethylnorspermine or BE-333). Mi et al.(1988) Prostate 34:51-60. While the precise role(s) ofnaturally-produced polyamines have not been clearly defined,interactions with DNA and RNA have been convincingly implicated. Sincethe nature of these interactions is highly structure-dependent,polyamine analogs have been designed to effectively disrupt polyaminefunction by competition with naturally-occurring polyamines. Severalpolyamine analogs have been developed that exert marked inhibition ofhuman tumor cell growth both in culture and in nude mice xenografts.Polyamine analogs such as BE-4444[1,19-bis(ethylamino)-5,10,15-triazanonadecane], BE-373[N,N′-bis(3-ethylamino) propyl)-1,7-heptane diamine], and BE-333 areparticularly effective in inhibiting prostate xenograft tumors in nudemice. Zagaja et al. (1998) Cancer Chem. Pharm. 41:505-512; Jeffers etal. (1997) Cancer Chem. Pharm. 40:172-179; Feuerstein et al. (1991) J.Cell. Biochem. 46:37-47; and Marton et al. (1995) Ann. Rev. Pharm.Toxicol. 35:55-91. However, polyamine analogs can cause systemictoxicity. BE-333, for example, causes side effects such as headache,nausea and vomiting, unilateral weakness, dysphagia, dysarthria,numbness, paresthesias, and ataxia. Creaven et al. (1997) Invest. NewDrugs 15:227-34. In one test, administration of BE-333 caused laboredbreathing, convulsive movements and acute death in rats. Kanter et al.(1994) Anticancer Drugs 5:448-56. This toxicity limits many polyamineanalogs to a small therapeutic window.

None of the above techniques for treating prostate diseases has beenuniversally successful. Following localized therapy, up to 40% ofpatients with advanced disease, and a large proportion of all patients,eventually develop metastatic disease. Treatment for advanced diseaseinitially involving hormonal manipulations and palliative radiotherapyhave demonstrated symptomatic relief, but not long-term disease-freesurvival. The use of cytotoxic agents in the management ofhormone-resistant advanced prostate cancer remains poorly defined. A fewsingle agents have become “standard therapy”, although demonstration oftheir efficacy, by contemporary standards, is lacking. Combinationalchemotherapy is frequently employed, although its contribution tooverall patient management is largely unsubstantiated, especially whencritical assessment of efficacy parameters are used. Newer approachesusing chemohormonal therapy and hormonal priming therapies have failed.High-dose chemotherapy with transplant regimens are not well-toleratedin an elderly population, to which most victims of prostate cancerbelong. A growth factor inhibitor, suramin, has shown promising initialresults, but also many side effects. Allolio et al. (1989) Dtsch. Med.Woschenschr. 114:381-4; and Broder et al. (1985) Lancet 2:627-30.However, no therapy to date has been demonstrated to improve overallsurvival in patients with advanced hormone refractory prostate cancer.

Approximately one out of every four males above the age of 55 suffersfrom a prostate disease. Due to the aging U.S. population, the incidenceof BPH, prostatitis and prostate cancer is likely to increase and tobecome an even more severe problem.

It would be advantageous to develop a new treatment of prostate cancerwhich retains the potency of chemotherapy without being subject to thevarious side effects and disadvantages of current therapies.

All references cited herein are hereby incorporated by reference intheir entirety.

SUMMARY OF THE INVENTION

The invention provides therapeutic compositions in which a cytostatic orcytocidal agent is conjugated to a polypeptide, where the polypeptide iscleaved from the agent by an enzyme.

In one embodiment, the cytostatic or cytocidal agent is a polyamineanalog. The polyamine analog can be linked to the peptide at the carboxyterminus of the peptide by an amide linkage to a primary or secondaryamine group of the polyamine. The polyamine analog can contain a hydroxygroup, and can be linked to the peptide at the carboxy terminus of thepeptide by an ester linkage through the hydroxy group. In anotherembodiment, the polyamine analog is conformationally restricted.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:—N(—E)—B—A—B—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH—E or—HN—B—A—B—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH—E

-   -   wherein each A is independently selected from the group        consisting of a single bond, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each B is        independently selected from the group consisting of: a single        bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E is        independently selected from the group consisting of H, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆        cycloaryl, and C₃-C₆ cycloalkenyl; and any salt or stereoisomer        thereof

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:—N(—E)—B—A—B—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH—E or—HN—B—A—B—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH—E

-   -   wherein each A is independently selected from the group        consisting of a single bond, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each B is        independently selected from the group consisting of: a single        bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E is        independently selected from the group consisting of H, C₁ -C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆        cycloaryl, and C₃-C₆ cycloalkenyl; with the proviso that either        at least one A moiety is selected from the group consisting of        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl,        and C₃-C₆ cycloalkenyl, or at least one B moiety is selected        from the group consisting of C₂-C₆ alkenyl; and any salt or        stereoisomer thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:—N(E)—B—A—B—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH (—B—A—B—NH)_(x)—E or—HN—B—A—B—NH—B—A—B—NH—B—A—B—NH(—B—A—B—NH)_(x)—E

-   -   wherein each A is independently selected from the group        consisting of: a single bond, C₆-C₂ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆        cycloalkenyl; each B is independently selected from the group        consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;        each E is independently selected from the group consisting of H,        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,        C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; and x is an integer        from 2 to 16; and any salt or stereoisomer thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:—N(—E)—B—A—B—NH—B—A—B—NH—B—A—B—NH(—B—A—B—NH)_(x)—E or—HN—B—A—B—NH—B—A—B—NH—B—A—B—NH(—B—A—B—NH)_(x)—E

-   -   wherein each A is independently selected from the group        consisting of: a single bond, C₆-C₂ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆        cycloalkenyl; each B is independently selected from the group        consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;        each E is independently selected from the group consisting of H,        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,        C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; and x is an integer        from 2 to 16; with the proviso that either at least one A moiety        is selected from the group consisting of C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆        cycloalkenyl, or at least one B moiety is selected from the        group consisting of C₂-C₆ alkenyl; and any salt or stereoisomer        thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:E—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH(—B—A—B—NH)_(x)—E

-   -   wherein each A is independently selected from the group        consisting of: a single bond, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each        B is independently selected from the group consisting of: a        single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E is        independently selected from the group consisting of C₁-C₆ alkyl,        C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, and        the peptide is linked to the polyamine via an ester linkage at        one and only one E group hydroxy; and x is an integer from 0 to        16; and any salt or stereoisomer thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula:E—NH—B—A—B—NH—B—A—B—NH—B—A—B—NH(—B—A—B—NH)_(x)—E

-   -   wherein each A is independently selected from the group        consisting of: a single bond, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each        B is independently selected from the group consisting of: a        single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; each E is        independently selected from the group consisting of C₁-C₆ alkyl,        C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆ hydroxyaryl, with        the proviso that at least one E moiety be selected from the        group consisting of C₁-C₆ alkanol, C₃-C₆ cycloalkanol, and C₃-C₆        hydroxyaryl, and the peptide is linked to the polyamine via an        ester linkage at one and only one E group hydroxy; and x is an        integer from 0 to 16; and any salt or stereoisomer thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula: —N(—E)—D—NH—B—A—B—NH—D—NH—E or—NH—D—NH—B—A—B—NH—D—NH—E

-   -   wherein A is selected from the group consisting of C₂-C₆        alkynyl; each B is independently selected from the group        consisting of: a single bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl;        each D is independently selected from the group consisting of        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,        C₃-C₆ cycloalkenyl, and C₃-C₆ cycloaryl; and each E is        independently selected from the group consisting of H, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆        cycloaryl, and C₃-C₆ cycloalkenyl; and any salt or stereoisomer        thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula: —N(—E)—B—A—B—NH—F—NH—B—A—B—NH—E or—NH—B—A—B—NH—F—NH—B—A—B—NH—E

-   -   wherein F is selected from the group consisting of C₁-C₆ alkyl;        each A is independently selected from the group consisting of: a        single bond, C₁-C₆ alkyl; C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each B is        independently selected from the group consisting of: a single        bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E is        independently selected from the group consisting of H, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆        cycloaryl, and C₃-C₆ cycloalkenyl; and any salt or stereoisomer        thereof.

In another embodiment of the invention, the polyamine analog linked tothe polypeptide is of the formula: —N(—E)—B—A—B—NH—F—NH—B—A—B—NH—E or—NH—B—A—B—NH—F—NH—B—A—B—NH—E

-   -   wherein F is selected from the group consisting of C₁-C₆ alkyl;        each A is independently selected from the group consisting of: a        single bond, C₁-C₆ alkyl; C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₃-C₆ cycloaryl, and C₃-C₆ cycloalkenyl; each B is        independently selected from the group consisting of: a single        bond, C₁-C₆ alkyl, and C₂-C₆ alkenyl; and each E is        independently selected from the group consisting of H, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆        cycloaryl, and C₃-C₆ cycloalkenyl; with the proviso that either        at least one A moiety is selected from the group consisting of        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloaryl,        and C₃-C₆ cycloalkenyl, or at least one B moiety is selected        from the group consisting of C₂-C₆ alkenyl; and any salt or        stereoisomer thereof.

In another embodiment, the cytostatic or cytocidal agent is a quinone,such as a naphthoquinone. In one embodiment, the naphthoquinone containsa hydroxy group and is linked to the peptide by the hydroxy group. Inanother embodiment, the naphthoquinone contains a primary or secondaryamino group and is linked to the peptide by the amino group.

In another embodiment of the invention, the quinone linked to thepolypeptide is selected from compounds of the formula

-   -   wherein A is —CH2-, —O—, —C(═O)—O—, or —O—C(═O)—, and M₁ is        C₁-C₈ alkyl, C₁-C₈ branched alkyl, C₃-C₈ cycloalkyl, or C₃-C₈        cycloaryl.

In another embodiment of the invention, the quinone linked to thepolypeptide is selected from compounds of the formula

-   -   wherein x is 1 or 2; and each K is independently selected from        the group consisting of H, OH, C₁-C₈ alkyl, C₁-C₈ alkenyl, C₁-C₈        alkanol, C₁-C₈ alkoxy, and    -   with the proviso that one and only one K is selected from the        group consisting of OH and C₁-C₈ alkanol, the peptide being        conjugated to the terminal hydroxy group of the alcohol; and        where zero or two, but no more than two, vicinal K's in the        molecule represent single electrons which form a pi bond, thus        forming a double bond together with the existing sigma bond        between the two adjacent carbons bearing the two vicinal K's.

In another embodiment of the invention, the quinone linked to thepolypeptide is selected from compounds of the formula

-   -   wherein Y is selected from the group consisting of —H, —F, —Br,        —Cl, and —I; and wherein G₁ is selected from the group        consisting of H, C₁-C₈ alkyl,        and —C(═O)—CH_(n)X_(3-n), where n is an integer from 0 to 3 and        X is selected from the group consisting of F, Cl, Br, and I; and        the peptide is conjugated to the quinone via the amino group        bearing G1.

In another embodiment of the invention, the quinone linked to thepolypeptide is selected from compounds of the formula

-   -   wherein x is 1 or 2; and each K is independently selected from        the group consisting of H, C₁-C₈ alkyl, C₁-C₈ alkenyl, C₁-C₈        alkoxy, and    -   and where zero or two, but no more than two, vicinal K's in the        molecule represent single electrons which form a pi bond, thus        forming a double bond together with the existing sigma bond        between the two adjacent carbons bearing the two vicinal K's.

In one embodiment of the invention, the cytostatic or cytocidal agent isconjugated to a polypeptide recognized and cleaved by prostate-specificantigen (PSA). In one embodiment, the polypeptide is recognized andcleaved by PSA and comprises less than about 25 amino acids. Preferably,the polypeptide comprises less than about 10 amino acids. Morepreferably, the polypeptide comprises the sequence HSSKLQ. Morepreferably, the polypeptide comprises or consists of the sequenceSKLQ-β-alanine or SKLQL, or comprises or consists of the sequence SKLQ.

In another embodiment, the cytostatic or cytocidal agent is conjugatedto a polypeptide recognized and cleaved by cathepsin B. In oneembodiment, the peptide sequence is X—P2-P1, where X is hydrogen, anamino-protecting group, or an amino-capping group attached to theN-terminus of P2; where P2 is the N-terminal amino acid and P1 is theC-terminal amino acid; and where P2 is a hydrophobic amino acid and P1is a basic or polar amino acid. In another embodiment, the peptidesequence is X—P2-P1-Y, where X is hydrogen, an amino-protecting group,or an amino-capping group attached to the N-terminus of P2; P2 is ahydrophobic amino acid; P1 is a basic or polar amino acid; and where Yis leucine, β-alanine, or a nonentity. In a further embodiment, X is a4-morpholinocarbonyl group. In yet another embodiment, P2 is selectedfrom the group consisting of leucine, isoleucine, valine, methionine,and phenylalanine; and P1 is selected from the group consisting oflysine, arginine, glutamine, asparagine, histidine and citrulline.

The invention also comprises compositions where the cytostatic orcytocidal agent conjugated to a polypeptide is combined with apharmaceutically acceptable excipient.

The invention also provides methods of treating cancers and otherdiseases characterized by cell proliferation, for example prostatecancer, in an individual comprising administering to the individual aneffective amount of a composition comprising a therapeutic amount of acytostatic or cytocidal agent conjugated to a polypeptide. Theseconjugates include polyamine analog conjugates or quinone conjugates ofthe present invention, for example, a polyamine analog or a quinoneconjugated to a polypeptide recognized and cleaved by an enzyme such asprostate-specific antigen (PSA) or cathepsin B. The disease can beprostatitis, benign prostate hyperplasia (BPH), or prostate cancer, andcan include suppression of the proliferation of metastatic tumors. Theindividual can be a mammal, and is preferably a human.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122 (□), SL-11123 (▪),SL-11126 (Δ), SL-11127 (▴), SL-11128 (⋄), SL-11129 (♦), SL-11130 (∇),SL-11133 (▾) on the survival of cultured human prostate cancer cellsPC3.

-   -   ED₅₀ of BE-4444=0.6 μM, SL-11121=0.52 μM, SL-11122>31.25 μM,        SL-11123>31.25 μM, SL-11126=0.2 μM SL-11127>31.25 μM,        SL-11128=0.5 μM, SL-11129=1.7 μM, SL-11130>31.25 μM, and        SL-11133>31.25 μM.

FIG. 2 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122 (□), SL-11123 (▪),SL-11126 (Δ), SL-11127 (▴), SL-11128 (⋄), SL-11129 (♦), SL-11130 (∇),and SL-11133 (▾) on the survival of cultured human prostate cancer cellsDU145.

-   -   ED₅₀ of BE-4444=0.07 μM, SL-11121=0.08 μM, SL-11122=0.08 μM,        SL-11123=0.51 μM, SL-11126=0.51 μM SL-11127 0.22 μM,        SL-11128=0.14 μM, SL-11129=0.32 μM, SL-11130=0.43 μM, and        SL-11133=0.34 μM.

FIG. 3 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122 (□), SL-11123 (▪),SL-11126 (Δ), SL-11127 (▴), SL-11128 (⋄), SL-11129 (♦), SL-11130 (∇),and SL-11133 (▾) on the survival of cultured human prostate cancer cellsDUPRO.

-   -   ED₅₀ of BE-4444=0.2 μM, SL-11121=0.4 μM, SL-11122=0.56 μM,        SL-11123>31.25 μM, SL-11126=1.1 μM, SL-11127 1.3 μM,        SL-11128=1.28 μM, SL-11129>31.25 μM, SL-11130>31.25 μM, and        SL-11133=31.25 μM.

FIG. 4 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●),SL-11126 (Δ), SL-11128 (⋄),on the survival of cultured human prostate cancer cells LNCAP.

-   -   ED₅₀ of BE-4444=0.14 μM, SL-11121=0.14 μM, SL-11126=0.55 μM and        SL-11128=0.3 μM.

FIG. 5 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122 (□), SL-11123 (▪),SL-11126 (Δ), SL-11127 (▴), and SL-11128 (⋄) on the survival of culturedhuman colon cancer cells HT29.

-   -   ED₅₀ of BE-4444=0.5 μM, SL-11121=0.8 μM, SL-11122=0.8 μM,        SL-11123=10.42 μM, SL-11126=1.5 μM, SL-11127=2.91 μM, and        SL-11128=1.35 μM.

FIG. 6 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122(□), SL-11123 (▪),and SL-11126(Δ) on the survival of cultured human lung cancer cellsA549.

-   -   ED₅₀ of BE-4444>31.25 μM, SL-11121>31.25 μM, SL-11122>31.25 μM,        SL-11123>31.25 μM, and SL-11126>31.25 μM.

FIG. 7 is a graph depicting the in vitro effect of increasingconcentrations of BE-4444 (◯), SL-11121 (●), SL-11122 (□), SL-11123 (▪),and SL-11126 (Δ) on the survival of cultured human breast cancer cellsMCF7.

-   -   ED₅₀ of BE-4444>31.25 μM, SL-11121=17.0 μM, SL-11122>31.25 μM,        SL-11123>31.25 μM, and SL-11126=0.7 μM.

FIG. 8 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), SL-11132 (▪), and BE-333(□) on the survival of cultured human prostate cancer cells PC3.

-   -   ED₅₀ of SL-11105>31.25 μM, SL-11124>31.25 μM, SL-11132>31.25 μM        and BE-333=0.34 μM.

FIG. 9 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), SL-11132 (▪), and BE-333(□) on the survival of cultured human prostate cancer cells DU145.

-   -   ED₅₀ of SL-11105=1.6 μM, SL-11124>31.25 μM, SL-11132=0.015 μM        and BE-333=0.12 μM.

FIG. 10 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), SL-11132 (▪), and BE-333(□) on the survival of cultured human prostate cancer cells DUPRO.

-   -   ED₅₀ of SL-11105=0.43 μM, SL-11124>31.25 μM, SL-11132>31.25 μM        and BE-333=0.9 μM.

FIG. 11 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), and BE-333 (□) on thesurvival of cultured human colon cancer cells HT29.

-   -   ED₅₀ of SL-11105=25.2 μM, SL-11124>31.25 μM, and BE-333=0.3 μM.

FIG. 12 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), and BE-333 (□) on thesurvival of cultured human lung cancer cells A549.

-   -   ED₅₀ of SL-11105=0.43 μM, SL-11124>31.25 μM, and BE-333=0.3 μM.

FIG. 13 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●), SL-11124 (◯), and BE-333 (□) on thesurvival of cultured human breast cancer cells MCF7.

-   -   ED₅₀ of SL-11105>31.25 μM, SL-11124>31.25 μM, and BE-333=3.7 μM.

FIG. 14 is a graph depicting the in vitro effect of increasingconcentrations of SL-11105 (●) and BE-333 (□) on the survival ofcultured human brain tumor cells U251 MG NCI.

-   -   ED₅₀ of SL-11105=25.9 μM, and BE-333=0.23 μM.

FIG. 15A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-1100 (□),SL-11101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival of culturedhuman prostate cancer cells PC3.

-   -   ED₅₀ of SL-11093=1.6 μM, SL-11098=1.4 μM, SL-11099=2.5 μM,        SL-1100=4.7 μM, SL-11101=7.7 μM, SL-11102>31.25 μM and        BE-444=0.7 μM.

FIG. 15B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), SL-11114(□), SL-11118 (▴), SL-11119 (Δ), and BE-444 (♦) on the survival ofcultured human prostate cancer cells PC3.

-   -   ED₅₀of SL-11103>31.25 μM, SL-11104>31.25 μM, SL-11108=2.2 μM,        SL-11114=0.7 μM, SL-11118=1.65 μM, SL-11119>31.25 μM and        BE-444=0.7 μM

FIG. 16A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-1100 (□),SL-1101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival of culturedhuman prostate cancer cells DU145.

-   -   ED₅₀ of SL-11093=0.016 μM, SL-11098=0.02 μM, SL-11099=0.014 μM,        SL-11100=0.021 μM, SL-11101=0.22 μM, SL-11102=0.03 μM and        BE-444=0.03 μM.

FIG. 16B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), SL-11114(□), SL-11118 (▴), SL-11119 (Δ), and BE-444 (♦) on the survival ofcultured human prostate cancer cells DU145.

-   -   ED₅₀ of SL-11103=2.8 μM, SL-11104=9.4 μM, SL-11108=0.13 μM,        SL-11114=0.13 μM, SL-11118=0.05 μM, SL-11119 0.08 μM and        BE-444=0.03 μM.

FIG. 17A is a graph depicting the in vitro effect of increasingconcentrations of SL-11099 (▪), SL-1100 (□), SL-11101 (▴), SL-11102 (Δ),and BE-444 (♦) on the survival of cultured human prostate cancer cellsDUPRO.

-   -   ED₅₀ of =SL-11099=0.08 μM, SL-11100=0.3 μM, SL-1101=0.85 μM,        SL-11102=0.15 μM and BE-444=0.2 μM.

FIG. 17B is a graph depicting the in vitro effect of increasingconcentrations of SL-11108 (▪), SL-11114 (□), SL-11118 (▴), SL-11119(Δ), and BE-444 (♦) on the survival of cultured human prostate cancercells DUPRO.

-   -   ED₅₀ of SL-11108=0.98 μM, SL-11114=0.64 μM, SL-11118=0.25 μM,        SL-11119=0.44 μM and BE-444=0.2 μM.

FIG. 18A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-11100(□), and BE-444 (♦) on the survival of cultured human prostate cancercells LNCAP.

-   -   ED₅₀ of SL-11093=0.21 μM, SL-11098=0.17 μM, SL-11099=0.21 μM,        SL-11100=0.7 μM, and BE-444=0.1 μM.

FIG. 18B is a graph depicting the in vitro effect of increasingconcentrations of SL-11108 (▪), SL-11114 (□), SL-11118 (▴), and BE-444(♦) on the survival of cultured human prostate cancer cells LNCAP.

-   -   ED₅₀ of SL-11108=7.7 μM, SL-11114=3.0 μM, SL-11118=0.21 μM, and        BE-444=0.1 μM.

FIG. 19A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-11110(□), SL-11101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival ofcultured human colon cancer cells HT29.

-   -   ED₅₀ of SL-11093=0.4 μM, SL-11098=0.4 μM, SL-11099=1.0 μM,        SL-11100=2.0 μM, SL-11101=5.2 μM. SL-11102=0.73 μM and        BE-444=0.93 μM.

FIG. 19B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), SL-11114(□), SL-11118 (▴), SL-11119 (Δ), and BE-444 (♦) on the survival ofcultured human colon cancer cells HT29.

-   -   ED₅₀ of SL-11103=29.4 μM, SL-11104=25.8 μM, SL-11108=2.0 μM,        SL-11114=3.6 μM, SL-11118=0.98 μM, SL-11119=0.97 μM and        BE-444=0.93 μM.

FIG. 20A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-11100(□), SL-11101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival ofcultured human lung cancer cells A549.

-   -   ED₅₀ of SL-11093=0.26 μM, SL-11098=0.29 μM, SL-11099=0.51 μM,        SL-11100=0.65 μM, SL-11101=2.2 μM, SL-11102=0.15 μM and        BE-444=0.15 μM.

FIG. 20B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), SL-11114(□),SL-11118 (▴), and BE-444 (♦) on the survival of cultured human lungcancer cells A549.

-   -   ED₅₀ of SL-11103=12.4 μM, SL-11104>31.25 μM, SL-11108>31.25 μM,        SL-1114>31.25 μM, SL-11118=0.214 μM and BE-444=0.15 μM.

FIG. 21A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098(◯), SL-11099(▪), SL-11100(□),SL-11101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival of culturedhuman breast cancer cells MCF7.

-   -   ED₅₀ of SL-11093=0.66 μM, SL-11098>31.25 μM, SL-11099=26.3 μM,        SL-11100>31.25 μM, SL-11101>31.25 μM SL-11102>31.25 μM and        BE-444>31.25 μM.

FIG. 21B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), and BE-444(♦) on the survival of cultured human breast cancer cells MCF7.

-   -   ED₅₀ of SL-11103>31.25 μM, SL-11104>31.25 μM, SL-11108>31.25 μM,        and BE-444>31.25 μM.

FIG. 22A is a graph depicting the in vitro effect of increasingconcentrations of SL-11093 (●), SL-11098 (◯), SL-11099 (▪), SL-11100(□), SL-11101 (▴), SL-11102 (Δ), and BE-444 (♦) on the survival ofcultured human brain tumor cells U251 MG NCI.

-   -   ED₅₀ of SL-11093=0.07 μM, SL-11098=0.1 μM, SL-11099=0.11 μM,        SL-11100=0.22 μM, SL-11101=1.7 μM, SL-11102=0.15 μM and        BE-444=0.2 μM.

FIG. 22B is a graph depicting the in vitro effect of increasingconcentrations of SL-11103 (●), SL-11104 (◯), SL-11108 (▪), and BE-444(♦) on the survival of cultured human brain tumor cells U251 MG NCI.

-   -   ED₅₀ of SL-11103=9.5 μM, SL-11104=14.71 μM, SL-11108=2.0 μM, and        BE-444=0.2 μM.

FIG. 23 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (▪) on thesurvival of cultured human prostate cancer cells PC3.

-   -   ED₅₀ of SL-11091>31.25 μM, SL-11094>31.25 μM, and BE-343=0.24        μM.

FIG. 24 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (▪) on thesurvival of cultured human prostate cancer cells DU145.

-   -   ED₅₀ of SL-11091=4.33 μM, SL-11094=15.4 μM, and BE-343=0.044 μM.

FIG. 25 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (▪) on thesurvival of cultured human colon cancer cells HT29.

-   -   ED₅₀ of SL-11091>31.25 μM, SL-11094=28.8 μM, and BE-343=0.6 μM.

FIG. 26 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (▪) on thesurvival of cultured human lung cancer cells A549.

-   -   ED₅₀ of SL-11091>31.25 μM, SL-11094>31.25 μM, and BE-343=0.2 μM.

FIG. 27 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (♦) on thesurvival of cultured human breast cancer cells MCF7.

-   -   ED₅₀ of SL-11091>31.25 μM, SL-11094>31.25 μM, and BE-343=0.5 μM.

FIG. 28 is a graph depicting the in vitro effect of increasingconcentrations of SL-11091 (●), SL-11094 (◯), and BE-343 (▪) on thesurvival of cultured human brain tumor cells U251 MG NCI.

-   -   ED₅₀ of SL-11091>31.25 μM, SL-11094>31.25 μM, and BE-343=0.14        μM.

FIG. 29 is a graph depicting the in vitro effect of increasingconcentrations of SL-11141 (●), SL-11144 (□), SL-11150 (▪) on thesurvival of cultured human prostate cancer cells PC3.

-   -   ED₅₀ of SL-11141>31.25 μM, SL-11144=0.3 μM, and SL-11150=0.5 μM.

FIG. 30 is a graph depicting the in vitro effect of increasingconcentrations of SL-11141 (●), SL-11144 (□), SL-11150 (▪) on thesurvival of cultured human prostate cancer cells DU145.

-   -   ED₅₀ of SL-11141=0.13 μM, SL-11144=0.1 μM, and SL-11150=0.11 μM.

FIG. 31 is a graph depicting the in vitro effect of increasingconcentrations of SL-11141 (●), SL-11144 (□), SL-11150 (▪) on thesurvival of cultured human prostate cancer cells DUPRO.

-   -   ED₅₀ of SL-11141=0.71 μM, SL-11144=0.36 μM, and SL-11150=0.48        μM.

FIG. 32 is a graph depicting the in vitro effect of increasingconcentrations of SL-11141 (●), SL-11144 (□), SL-11150 (▪) on thesurvival of cultured human prostate cancer cells LNCAP.

-   -   ED₅₀ of SL-11141=0.07 μM, SL-11144=0.20 μM, and SL-11150=0.23        μM.

FIG. 33 illustrates synthetic methodology used to prepare polyaminecompounds useful in the invention.

FIG. 34 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 35 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 36 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 37 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 38 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 39 illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 40A illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 40B illustrates additional synthetic methodology used to preparepolyamine compounds useful in the invention.

FIG. 41 depicts Scheme 501, illustrating the synthetic preparation ofquinone compounds useful in the invention.

FIG. 42 depicts Scheme 502, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 43 depicts Scheme 503, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 44 depicts Scheme 504, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 45 depicts Scheme 505, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 46 depicts Scheme 506, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 47 depicts Scheme 507, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 48 depicts Scheme 508, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 49 depicts Scheme 509, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 50 depicts Scheme 510, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 51 depicts Scheme 511, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 52 depicts Scheme 512, illustrating the synthetic preparation ofadditional quinone compounds useful in the invention.

FIG. 53 depicts Scheme 513, illustrating synthetic preparation ofpeptides conjugated to certain quinone compounds.

FIG. 54 depicts Scheme 514, illustrating additional syntheticpreparation of peptides conjugated to certain quinone compounds.

FIG. 55 depicts Scheme 25, illustrating the synthesis of a dipeptideconjugated to a polyamine alchohol via an ester linkage.

FIG. 56 depicts Scheme 26, illustrating the synthesis of a tetrapeptideconjugated to various polyamines via an amide linkage.

FIG. 57 is a graph depicting the in vitro effects of increasingconcentrations of SL-11141 (◯) and SL-11155 (●) on the survival ofcultured human prostate cancer cells PC3.

FIG. 58 is a graph depicting the in vitro effects of increasingconcentrations of SL-11141 (◯) and SL-11155 (●) on the survival ofcultured human prostate cancer cells DUPRO.

FIG. 59 is a graph depicting the in vitro effects of increasingconcentrations of SL-11141 (◯) and SL-11155 (●) on the survival ofcultured human prostate cancer cells LNCAP.

FIG. 60 is a graph depicting the in vitro effects of increasingconcentrations of SL-11141 (●) and BE 4×4 (◯) and SL-11155 on thesurvival of cultured human prostate cancer cells DU145.

MODES FOR CARRYING OUT THE INVENTION

The present invention encompasses polyamine analog and quinoneconjugates in which a polyamine analog or a quinone is conjugated to apolypeptide to form an inactive prodrug. The conjugates of the inventionare useful in treating cancers and other diseases characterized by cellproliferation. The polyamine analog conjugates of the present inventionare particularly useful in suppression of proliferation of prostatecells.

The polypeptides are preferably enzyme substrates, designed to bespecifically recognized and cleaved by enzymes overexpressed by, orexpressed exclusively by, cancerous cells or cells of the target tissue.Prostate specific antigen (PSA), for example, is produced in largeamounts only by prostate tissues. A peptide substrate for PSA can thusbe bound to a polyamine analog to form a prodrug. When the polypeptidemoiety of the conjugate is removed by PSA, the prodrug becomes activeand the polyamine analog can inhibit proliferation of the prostate cell.This proliferation inhibition is useful in treating a variety ofprostatic diseases.

PSA is a protease expressed in the highly specializedapically-superficial layer of secretory (luminal) cells of the prostategland, as well as at other sites of the urogenital tract, frequentlycoinciding with glucosamine glucans, glycoproteins and numerous enzymeproteins. PSA is found in seminal fluid in its free form and in serum,where it occurs in an inactive complex form with alpha₁-chymotrypsin.PSA has chymotrypsin-like substrate specificity. Lilja et al. (1985) J.Clin. Invest. 76:1899-1903; Watt et al. (1986) Proc. Natl. Acad. Sci.USA 83:3166-3170; and Christensson et al. (1990) J. Biochem.194:755-765. PSA specifically recognizes and cleaves polypeptides,including those of sequences HSSKLQ and SKLQ, which are not recognizedby abundant serum proteases. Denmeade et al. (1997) Cancer Res.57:4924-30; and Denmeade et al. (1998) Cancer Res. 58:2537-40. Whileboth normal and cancerous prostate tissues produce PSA [Denmeade et al.(1997)], PSA levels in the seminal fluid and blood serum increasemany-fold in patients with prostate tumors. Increased PSA levels arealso detected in patients with BPH or prostatitis. Rainwater et al.(1990) Mayo Clinic Proc. 65:11118-26. In addition, even when blood serumPSA levels increase up to 1000 ng/ml in patients with advanced prostatecancer, PSA in the blood serum is inactive. Denmeade et al. (1997).

Conjugation of the polyamine analogs with a polypeptide cleaved by PSAdecreases the danger of toxicity of the polyamine analog in two ways.First, the polypeptide moiety reduces biological activity of thepolyamine analog outside of the target tissues. Second, because thepolypeptides are recognized by PSA and thus target the prodrug to theprostate, a lower dosage of polyamine analog can be administered. Asdiscussed below, the polyamine analog can be any polyamine analog,including, but not limited to, 1, 12-Me₂-SPM, SL-11027, SL-11028,SL-11029, SL-11033, SL-11034, SL-11037, SL-11038, SL-11043, SL-11044,SL-11047, SL-11048, SL-11050, SL-11090, SL-11091, SL-11092, SL-11093,SL-11094, SL-11098, SL-11099, SL-11100, SL-11101, SL-11102, SL-11103,SL-11104, SL-11105, SL-11108, SL-11114, SL-11118, SL-11119, SL-11121,SL-11122, SL-11123, SL-11124, SL-11126, SL-11127, SL-11128, SL-11129,SL-11130, SL-11132, SL-11133, SL-11134, SL-11136, SL-11137, SL-11141,SL-11144, SL-11150, SL-11201, and SL-11202. Preferably, the polyamineanalog is conformationally restricted.

Definitions

By “polyamine analog” is meant an organic cation structurally similarbut non-identical to polyamines such as spermine and/or spermidine andtheir precursor, diamine putrescine. By a “polyamine” is meant any of agroup of aliphatic, straight-chain amines derived biosynthetically fromamino acids; polyamines are reviewed in Marton et al. (1995) Ann. Rev.Pharm. Toxicol. 35:55-91. Polyamines cadaverine and putrescine arediamines produced by decarboxylation of lysine or omithine,respectively. Putrescine is converted to spermidine, and spermidine tospermine, by the addition of an aminopropyl group. This group isprovided by decarboxylated S-adenosyl methionine. Polyamine analogs,which can be branched or un-branched, include, but are not limited to,BE-4444 [1,19-bis(ethylamino)-5,10,15-triazanonadecane]; BE-333[N1,N11-diethylnorspermine; DENSPM;1,11-bis(ethylamino)-4,8-diazaundecane; thermine; Warner-Parke-Davis];BE-33 [N1,N7-bis(ethyl)norspermidine]; BE-34[N1,N8-bis(ethyl)spermidine]; BE-44 [N1,N9-bis(ethyl)homospermidine];BE-343 [N1,N12-bis(ethyl)spermine; diethylspermine-N1-N12; DESPM];BE-373 [N,N′-bis(3-ethylamino)propyl)-1,7-heptane diamine, Merrell-Dow];BE-444 [N1,N14-bis(ethyl)homospermine; diethylhomospermine-N1-N14];BE-3443 [1,17-bis(ethylamino)-4,9,14-triazaheptadecane]; BE-4334[1,17-bis(ethylamino)-5,9,13-triazaheptadecane]; 1,12-Me₂-SPM[1,12-dimethylspermine]; various polyamine analogs disclosed in WO98/17624 and U.S. Pat. No. 5,889,061; and the various novel polyamineanalogs illustrated in the Figures and described herein, including, butnot limited to, compounds designated SL-11027, SL-11028, SL-11029,SL-11033, SL-11034, SL-11037, SL-11038, SL-11043, SL-11044, SL-11047,SL-11048, SL-11050, SL-11090, SL-11091, SL-11092, SL-11093, SL-11094,SL-11098, SL-11099, SL-11100, SL-11101, SL-11102, SL-11103, SL-11104,SL-11105, SL-11108, SL-11114, SL-11118, SL-11119, SL-11121, SL-11122,SL-11123, SL-11124, SL-11126, SL-11127, SL-11128, SL-11129, SL-11130,SL-11132, SL-11133, SL-11134, SL-11136, SL-11137, SL-11141, SL-11144,SL-11150, SL-11201, and SL-11202. Additional polyamine analogs usefulfor this invention are known in the art, such as O'Sullivan et al.(1997) Bioorg. Med. Chem. 5:2145-2155; and Mukhopadhyaya et al. (1995)Exp. Parasit. 81:39-46; and U.S. Pat. No. 4,935,449.

By “conformationally restricted” is meant that, in a polyamine analog,at least two amino groups are locked or limited in spatial configurationrelative to each other. The relative movement of two amino groups can berestricted, for example, by incorporation of a cyclic or unsaturatedmoiety between them (exemplified, but not limited to, a ring, such as athree-carbon ring, four-carbon ring, five-carbon-ring, six-carbon ring,or a double or triple bond, such as a double or triple carbon bond).Groups restricting conformational flexibility by means of sterichindrance, yet structurally favorable to the anti-proliferative effects,can also be used according to the invention. A “conformationallyrestricted” polyamine analog can comprise at least two amino groupswhich are conformationally restricted relative to each other, but canalso further comprise amino groups which are not conformationallyrestricted relative to each other. Flexible molecules such as spermineand BE-444 can have a myriad of conformations and are therefore notconformationally restricted.

By “cancer” is meant the abnormal presence of cells which exhibitrelatively autonomous growth, so that they exhibit an aberrant growthphenotype characterized by a significant loss of cell proliferationcontrol. One embodiment of the present invention comprises methods oftreating prostate cancer.

By “prostate” is meant the muscular, glandular organ which surrounds theurethra of males at the base of the bladder. The prostate is anon-essential organ.

For purposes of this invention, “PSA” includes anyfunctionally-preserved variant, derivative and/or fragment of PSA,including amino acid sequence variants and proteins differing inpost-translational modification, which retain the sequence-specificproteolytic ability of PSA.

For purposes of this invention, “cathepsin B” includes anyfunctionally-preserved variant, derivative and/or fragment of cathepsinB, including amino acid sequence variants and proteins differing inpost-translational modification, which retain the sequence-specificproteolytic ability of cathepsin B.

The terms “polypeptide”, “polypeptide moiety”, “protein”, and the likeare used interchangeably herein to refer to any polymer of amino acidresidues of any length. The polymer can be linear or non-linear (e.g.,branched), it can comprise modified amino acids or amino acid analogs,and it can be interrupted by chemical moieties other than amino acids.The terms also encompass an amino acid polymer that has been modifiednaturally or by intervention; for example, by disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as, conjugation with a labeling orbioactive component. The polypeptide components of the conjugates of thepresent invention are recognized and cleaved by enzymes such asprostate-specific antigen (PSA) or cathepsin B. Preferably, thespecificity of cleavage is such that the polypeptide is cleaved toproduce a free polyamine analog or free quinone with biological activityor a polyamine analog or free quinone with a very short residualpolypeptide or single amino acid attached, which residual polypeptide orsingle amino acid does not interfere with the desired biologicalactivity of the polyamine analog or quinone.

By “conjugation” is meant the process of forming a covalent linkage,with or without an intervening linker, between two moieties, such as apolyamine analog and a polypeptide moiety. The conjugation can beperformed by any method known in the art, such as those described inWong, Chemistry of Protein Conjugation and Cross-linking, 1991, CRCPress, Boca Raton, and described herein. Suitable methods include usingstrategies incorporating protecting groups such as thet-butyloxycarbonyl (BOC) protecting group (reagents for introducing theBOC group are available from Sigma, St. Louis, Mo., and othersuppliers). Other suitable protecting groups which can be used in theconjugation reactions are described in Greene et al., Protective Groupsin Organic Synthesis, 2nd Edition, 1991, Wiley, New York. Preferably,the polypeptide moiety is conjugated to the polyamine analog moiety orquinone moiety such that (1) the presence of the polypeptide moietyprevents the functionality of the polyamine analog or quinone; and (2)cleavage by an enzyme produces a free polyamine analog or free quinone,or a polyamine analog or quinone with such a small residual portion ofthe polypeptide moiety remaining attached, so that the polyamine analogis capable of effecting anti-proliferative activity. By “conjugate” ismeant a chemical entity comprising two moieties which are covalentlylinked.

An “amino-capping group” or “amino-terminal capping group” is a groupthat covalently links to an amino group. Examples of amino-cappinggroups include, but are not limited to, 4-morpholinocarbonyl, acetyl,and trifluoroacetyl. An “amino-protecting group” or “amino-terminalprotecting group” is a group that can be selectively removed from anamino group of a molecule without affecting the remainder of themolecule. Examples of amino-protecting groups include, but are notlimited to, t-butyloxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC),benzyloxycarbonyl (CBZ), t-butyldimethylsilyl (TBDIMS), or suitablephotolabile protecting groups such as 6-nitroveratryloxy carbonyl (Nvoc)and the like.

An “exterior nitrogen” or “exterior amino group” of a polyamine orpolyamine analog is a nitrogen (amino) group which is flanked by onlyone other nitrogen group, while an “interior nitrogen” or “interioramino group” of a polyamine or polyamine analog is a nitrogen (amino)group which is flanked by two other nitrogen (amino) groups. Forexample, in a polyamine of the formula R₁—N¹H—R₂—N²H—R₃—N³H— . . .—R_((n-1))—N^((n-1))H—N^(n)H—R_(n), where n is an integer, the nitrogensdesignated as N¹ and N^(n) are the “exterior nitrogens” or “exterioramino groups,” inasmuch as they are flanked by only one other nitrogengroup, while N², N³, etc., through N^((n-1)) are “interior nitrogens” or“interior amino groups,” flanked by two other nitrogen (amino) groups.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets. Preferably, the individual isknown or suspected to be afflicted by a prostate disease, such as BPH,prostatitis and/or prostate cancer. When the individual is not a human,a determination should be made of the specific amino acid sequencerecognized and cleaved by the PSA of that individual's species. Thepolypeptide moiety of the polyamine analog conjugate should be suitablymodified in sequence, if necessary, to be recognized and cleaved by thePSA present in that individual's species.

An “effective amount” or “therapeutic amount” is an amount sufficient toeffect beneficial or desired clinical results. An effective amount canbe administered in one or more administrations. For purposes of thisinvention, an effective amount of a polyamine analog conjugate is anamount that is sufficient to palliate, ameliorate, stabilize, reverse,slow or delay the progression of the disease state. A therapeutic amountof a polyamine analog conjugate of the present invention is an amountsufficient to inhibit proliferation of prostate cells. A polyamineanalog conjugate is considered to be an effective agent for treatingprostate diseases if it is effective against, for example, at least onetype of prostate cancer cell line, even if it is not effective against adifferent prostate cell line.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results, including, but not limited to, the suppressionof proliferation of prostate cells. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease,stabilization (i.e., not worsening) of state of disease, prevention ofspread (i.e., metastasis) of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state,improvement in quality of enjoyment of life, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment.

By “suppressing proliferation of prostate cells” means that theproliferation of cells of the prostate gland, prostate-derived tumorcells, including metastatic tumors, or any cells expressing PSA isinhibited.

“Palliating” a disease means that the extent and/or undesirable clinicalmanifestations of a disease state are lessened and/or time course of theprogression is slowed or lengthened, as compared to not administeringpolyamine analog conjugates of the present invention.

Polyamine Analogs Useful in the Invention

One embodiment of the present invention encompasses a polyamine analogconjugated to a polypeptide specifically recognized and cleaved byprostate-specific antigen (PSA). Another embodiment of the presentinvention encompasses a polyamine analog conjugated to a polypeptidespecifically recognized and cleaved by cathepsin B. Other aspects of theinvention encompass compositions comprising these conjugate(s). Thepolyamine analogs which may be used are as described below.

Generally, polyamine analog conjugates of the present invention can beproduced by the following procedure. First, a polyamine analog isselected or a novel polyamine analog is designed. Without wishing to bebound by any particular theory explaining polyamine analog toxicity, itis believed that design of a novel polyamine analog can be based on thecurrent knowledge of polyamine interaction with DNA and ability toinduce structural changes in nucleic acids. Feuerstein et al. (1991);Gosule et al. (1978) J. Mol. Biol. 121:311-326; Behe et al. (1981) Proc.Natl. Acad. Sci. USA 78:1619-23; Jain et al. (1989) Biochem.28:2360-2364; and Basu et al. (1990) Biochem. J. 269:329-334.Alternatively, a novel polyamine analog can be designed based on itslikely ability to inhibit cell growth by suppressing natural polyaminesynthesis or deplete the intracellular natural polyamine pool. Porter etal. (1988) in Advances in Enzyme Regulation, Pergamon Press, pp. 57-79.In the next step, the polyamine analog is tested in vitro for efficacyin inhibiting proliferation of prostate cells (such as LNCaP cells, PC-3cells, or DUPRO cells). If it is efficacious, the polyamine analog isconjugated to a polypeptide. The polyamine analog conjugate can then betested for its ability to be specifically recognized and cleaved by PSA,but not by other proteases, in a cell-free medium in vitro. If thepolyamine analog conjugate passes this test, it can then be tested inanimals, such as nude mice with prostate cancer xenografts. Testing canthen proceed to human trials.

Conformationally Restricted Polyamine Analogs

Any polyamine analog (which has the requisite functional cytostatic orcytocidal property) may be used that has a pendant amino or hydroxylgroup which can be conjugated to the C-terminus of the polypeptidemoiety in an amide linkage or ester linkage, respectively, and examplesare provided in the summary of the invention, the definition of“polyamine analogs” and in the synthetic schemes. Polyamine analogs usedin the present invention are preferably conformationally restricted.Conformation is a determinant of the spatial arrangement of thepharmacophore or functional groups which interact with receptor bindingsites. The latter prefer specific ligand conformations or a specificdistribution of conformations. A flexible molecule such as spermine orBE-4444 can have a myriad of conformations. The conformer that binds tothe macromolecule (e.g., DNA or RNA) may not necessarily be the one withthe lowest energy as determined by spectroscopic methods ortheoretically by molecular mechanics calculations. The binding energy ofthe polyamine analog binding to the nucleic acid may be overcome withformation of an unstable conformer. Conversely, in the presence of aconformationally rigid analog of a flexible molecule, the hostmacromolecule might change its overall conformation or the distancesfrom one strand to the other. Hydrogen bonding is the main binding forceof either spermine or spermidine associating with the helical region ofa tRNA, and very likely also with DNA. Frydman et al. (1992) Proc. Natl.Acad. Sci. USA 89:9186-9191; and Fernandez et al. (1994) Cell Mol. Biol.40: 933-944. The secondary amino groups present in the linear spermineanalogs BE-4444 are the groups most directly involved in the formationof the hydrogen bonds with the paired bases of tRNA. While not wishingto limit the invention to any particular theory of operation, it isbelieved that those amino groups that usually flank the centralfour-carbon or three-carbon segment of the polyamine analog are mostlikely to function as the pharmacophore. When the nitrogens areseparated by only a two-carbon segment they are not protonated at pH 7.4and hence they do not form hydrogen bonds. If these amino groups arelocked into various configurations by the incorporation of cyclic orunsaturated moieties into the polyamine analog molecule, aconformationally rigid analog is obtained. When such analogs bind to DNAor tRNA, they will very likely induce a change in the conformation ofthe nucleic acid strands or loops that may differ from theconformational changes induced by the natural polyamines.

Schemes 1-25 depict syntheses of various polyamine analogs which can beused in the invention. Examples of polyamine analogs which can be usedin the invention are also given in U.S. Pat. Nos. 5,889,061 and5,627,215, which describe tetraamino polyamine analogs. The synthesis ofthe polyamine analogs of those patents can be modified to introduce anamino-protecting group on the exterior nitrogens (i.e., representing thetetraamine as R₁—N¹H—R₂—N²H—R₃—N³H—R₄—N⁴H—R₅, the nitrogens designatedas N¹ and N⁴ are the “exterior” nitrogens, inasmuch as they are flankedby only one other nitrogen group, while N² and N³ are “interior”nitrogens, flanked by two other nitrogen groups) in place of the groupthat would ordinarily be attached at that point (in this example, aprotecting group would be used instead of R₁ or R₅), and can be cleavedto yield a primary amino group at one of the exterior nitrogens, whilemaintaining amino-protecting groups on the other exterior nitrogen andthe interior nitrogens. Scheme 26 depicts such a strategy ofestablishing a protecting group regimen which allows one of the exterioramino groups to be selectively deprotected, while maintaining theamino-protecting groups on the other exterior amino group and theinterior amino groups. Examples of differential protection regimens ofpolyamines are also given in Fiedler et al. (1993) Helv. Chim. Acta76:1511-1519 and Iwata et al. (1989) Bull. Chem. Soc. Japan62:1102-1106. TABLE 1 No. Structure SL-11027

SL-11028

SL-11029

SL-11033

SL-11034

SL-11035

SL-11036

SL-11037

SL-11038

SL-11043

SL-11044

SL-11047

SL-11048

SL-11050 BnNH(CH₂)₄NHBn.2HCl SL-11061EtNH(CH₂)₄NH(CH₂)₄NH(CH₂)₄NH(CH₂)₄—NHEt.5HCl SL-11090

SL-11091

SL-11092

SL-11093

SL-11094

SL-11098

SL-11099

SL-11100

SL-11101

SL-11102

SL-11103

SL-11104

SL-11105

SL-11108

SL-11114

SL-11118

SL-11119

SL-11121

SL-11122

SL-11123

SL-11124

SL-11126

SL-11127

SL-11128

SL-11129

SL-11130

SL-11132

SL-11133

SL-11134

SL-11135

SL-11136

SL-11137

SL-11141

SL-11143

SL-11144

SL-11150

SL-11155

SL-11157

SL-11158

SL-11159

SL-11160

Efficacy of Polyamine Analogs Against Tumor Cells In Vitro

Novel polyamine analogs, once designed and constructed, are tested forefficacy in vitro against disease cells, such as prostate tumor cells.Known polyamines can also be tested in this way. Analogs found to beactive against disease cells are particularly suitable for use in theconjugates and methods of the invention.

Quinones Useful in the Invention

Quinones useful in the invention include cytotoxic quinones with apendant amino or hydroxyl group which can be conjugated to theC-terminus of the polypeptide moiety in an amide linkage or esterlinkage, respectively. Syntheses of several quinones useful in theinvention are presented below. Additional examples of quinones useful inthe invention, along with methods for their preparation, are found inU.S. Pat. No. 5,763,625 (including, but not limited to, those compoundsdescribed at column 4, lines 40 to 52, where R is (CH₂)_(n)—R₁ and R₁ isa hydroxy or an amine); in U.S. Pat. No. 5,824,700 (including, but notlimited to, those compounds of formula II at column 4, lines 20 to 48,which contain amino or hydroxy groups), and in U.S. Pat. No. 5,883,270(including, but not limited to, those compounds of formula I at column1, lines 49 to 67, which contain amino groups).

Preparation of Polypeptide Moiety of the Conjugates of the Invention

The polypeptide moiety of the conjugates of the invention, such as thepolyamine analog conjugates or quinone conjugates of the presentinvention, should be specifically recognized and cleaved by enzymespresent at high levels in the target tissue relative to levels innon-targeted tissues, or enzymes more readily accessible in the targettissue relative to non-targeted tissue. One example of such an enzyme isprostate specific antigen, which is present in high levels in theprostate. Another example of such an enzyme is cathepsin B, which isnormally present primarily in lysosomes, but which has been found insome cancers to be associated with the extracellular face of the plasmamembrane (as well as being overexpressed in cancer cells relative tonormal cells). See Yan et al. (1998) Biol. Chem. 379:113-123. CathepsinB is believed to play a role in degradation of the extracellular matrix,facilitating angiogenesis by tumors. Sinha et al. (1995) Anat. Rec.241:353-362; Sinha et al. (1995) Prostate 26:171-178. Peptides attachedto doxorubicin are disclosed in Dubowchik et al. (1998) Bioorg. Med.Chem. Lett. 8:3341-3346.

For use in targeting prostate tissue, the polypeptide components of theconjugates of the present invention comprise less than about 100 aminoacids, preferably less than about 50 amino acids, more preferably lessthan about 25 amino acids, preferably less than about 10 amino acids,more preferably about seven or fewer amino acids, and most preferably,four or five amino acids. Preferably, the polypeptide comprises theamino acid sequence HSSKLQ, more preferably it comprises the sequence oftetrapeptide SKLQ, and even more preferably it consists of SKLQ. Inanother embodiment, the peptide comprises the sequence SKLQL orSKLQ-β-alanine, and in a more preferred embodiment, the peptide consistsof SKLQL or SKLQ-β-alanine. The peptide sequences can include N-terminalmodifications, including, but not limited to, capping with amino-cappinggroups such as 4-morpholinocarbonyl and acetyl, or protection withprotecting groups such as benzyloxycarbonyl (Cbz) or t-butyloxycarbonyl(Boc).

When cleavage of the polypeptide by cathepsin B is desired, the peptidewill generally comprise less than about 10 amino acids, preferably lessthan about 4 amino acids. In preferred embodiments, the peptidescomprise two or three amino acids. Preferred sequences includedipeptides of the sequence X—P2-P1, where P2 is the N-terminal aminoacid and P1 is the C-terminal amino acid, where X is hydrogen, anamino-protecting group, or an amino-capping group; P2 is a hydrophobicamino acid; and P1 is a basic or polar amino acid. Another preferredsequence includes tripeptides of the form X—P2-P1-β-alanine orX—P2-P1-leucine, where P2 is the N-terminal amino acid and β-alanine orleucine is the C-terminal amino acid. A preferred embodiment for X is4-morpholinocarbonyl. Preferred amino acids for P2 include leucine,isoleucine, valine, methionine, and phenylalanine. Preferred amino acidsfor P1 include lysine, arginine, glutamine, asparagine, histidine andcitrulline. The peptide sequences may include N-terminal modifications,including, but not limited to, capping with amino-capping groups such as4-morpholinocarbonyl and acetyl, or protection with protecting groupssuch as benzyloxycarbonyl (Cbz) or t-butyloxycarbonyl (Boc).

The polypeptides used in this invention can be made by procedures knownin the art. The polypeptides can be produced by recombinant methods(i.e., single or fusion polypeptides) or by chemical synthesis.Polypeptides, especially shorter polypeptides up to about 50 aminoacids, are conveniently made by chemical synthesis. See, for example,Atherton and Sheppard, Solid Phase Peptide Synthesis: A PracticalApproach, New York: IRL Press, 1989; Stewart and Young: Solid-PhasePeptide Synthesis 2nd Ed., Rockford, Ill.: Pierce Chemical Co., 1984;and Jones, The Chemical Synthesis of Peptides, Oxford: Clarendon Press,1994. The polypeptides can be produced by an automated polypeptidesynthesizer employing the solid phase method, such as those sold byPerkin Elmer-Applied Biosystems, Foster City, Calif., or can be made insolution by methods known in the art.

Polypeptides can also be made by expression systems, using recombinantmethods. The availability of polynucleotides encoding polypeptidespermits the construction of expression vectors encoding polypeptides. Apolynucleotide encoding the desired polypeptide, whether in fused ormature form, and whether or not containing a signal sequence to permitsecretion, may be ligated into expression vectors suitable for anyconvenient host. Both eukaryotic and prokaryotic host systems can beused. The polypeptide is then isolated from lysed cells or from theculture medium and purified to the extent needed for its intended use.Purification or isolation of the polypeptides expressed in host systemscan be accomplished by any method known in the art. For example, cDNAencoding a polypeptide intact or a fragment thereof can be operativelylinked to a suitable promoter, inserted into an expression vector, andtransfected into a suitable host cell. The host cell is then culturedunder conditions that allow transcription and translation to occur, andthe desired polypeptide is recovered. Other controlling transcription ortranslation segments, such as signal sequences that direct thepolypeptide to a specific cell compartment (i.e., for secretion), canalso be used. Examples of prokaryotic host cells are known in the artand include, for example, E. coli and B. subtilis. Examples ofeukaryotic host cells are known in the art and include yeast, avian,insect, plant, and animal cells such as COS7, HeLa, CHO and othermammalian cells.

A fusion protein may also be constructed that facilitates purification.Examples of components for these fusion proteins include, but are notlimited to myc, HA, FLAG, His-6, glutathione S-transferase, maltosebinding protein or the Fc portion of immunoglobulin. These methods areknown in the art. See, for example, Redd et al. (1997) J. Biol. Chem.272:11193-11197.

Preferably, especially if used for diagnostic purposes, the polypeptidesare at least partially purified or isolated from other cellularconstituents. Preferably, the polypeptides are at least 50% pure. Inthis context, purity is calculated as a weight percent of the totalprotein content of the preparation. More preferably, the proteins are50-75% pure. More highly purified polypeptides may also be obtained andare encompassed by the present invention. For clinical use, thepolypeptides are preferably highly purified, at least about 80% pure,and free of pyrogens and other contaminants. Methods of proteinpurification are known in the art and are not described in detailherein.

The polypeptide(s) must be cleavable by the enzyme targeted, such as PSAor cathepsin B. A polypeptide can be readily tested for thischaracteristic by determining whether cleavage has occurred when thepolypeptide(s) is reacted under suitable conditions with PSA orcathepsin B. See, e.g., Denmeade et al. (1997).

Conjugation of Polyamine Analogs and Quinones to the Polypeptide Moiety

Any method known in the art can be used to conjugate (i.e., link) thepolypeptide recognized and cleaved by enzymes such as PSA or cathepsin Bto the polyamine analog or quinone, including, but not limited to, thosedisclosed herein. Suitable methods include using strategiesincorporating protecting groups such as the t-butyloxycarbonyl (BOC)protecting group (reagents for introducing the BOC group are availablefrom Sigma, St. Louis, Mo., and other suppliers). Other suitableprotecting groups which can be used in the conjugation reactions aredescribed in Greene et al., Protective Groups in Organic Synthesis, 2ndEdition, 1991, Wiley, New York. Preferably, the polypeptide moiety isconjugated to the polyamine analog moiety or quinone moiety such that(1) the presence of the polypeptide moiety prevents the functionality ofthe polyamine analog or quinone; and (2) cleavage by PSA produces a freepolyamine analog or quinone, or a polyamine analog or quinone with sucha small residual portion of the polypeptide moiety remaining attached,so that the polyamine analog or quinone is capable of effectinganti-proliferative activity.

The peptides are preferentially coupled via the α-COOH group of theC-terminal amino acid, although other linkages are possible, dependingon the peptide sequence (e.g., the γ-carboxyl group of a glutamic acidresidue can be used for linkage). When a polyamine is coupled, thelinkage will be via an amino group of the polyamine (i.e., an amidelinkage); when a polyamine alcohol is conjugated, the linkage can be viaeither an amino group or a hydroxy group of the polyamine alcohol (i.e.,an amide linkage or ester linkage, respectively). When an amide linkageto a polyamine is used, the peptide is preferably coupled to an exteriornitrogen. When an ester linkage to a polyamine is used, the peptide ispreferably coupled to a terminal hydroxy group. When a quinonecontaining an amino group is conjugated, the linkage to the peptide willbe an amide linkage; when the quinone contains a hydroxy group, thelinkage will be an ester linkage; and when the quinone contains bothgroups, either an amide linkage or an ester linkage can be employed.

In Vitro and In Vivo Testing of Polyamine Analog and Quinone Conjugates

When a polyamine analog or quinone has been shown to be effective invitro, its conjugate can be constructed and also tested in vitro.Preferably, in vitro testing of polyamine analog or quinone conjugatesshould be performed with the same cell lines that demonstrated efficacyof the polyamine analogs or quinones themselves, e.g. human prostatecancer cell lines PC-3, DU-145 and DuPro. U.S. Pat. Nos. 5,883,270,5,889,061, 5,763,625, and 5,824,700 all provide examples of protocolsused to test compounds for biological activity.

Those conjugates shown to have efficacy in vitro are generally nexttested in vivo. Prostate tumor xenografts can be grown in nude mice, forexample, and polyamine analog conjugates or quinone conjugatesadministered to these test animals. Determination of efficacy caninclude measurement of effective dosage and monitoring of side effects.

Methods of Administration of Polyamine Analog Conjugates

The invention also provides methods of treatment and methods ofsuppressing cell proliferation or uncontrolled cell growth, especiallyprostate cell proliferation. The methods comprise administering aneffective amount of any of the conjugates described herein. Fortreatment, an effective amount is an amount sufficient to palliate,ameliorate, stabilize, reverse, slow or delay the disease state; or forinhibition or suppression of proliferation of cells such as prostatecells.

Polyamine analog conjugates of the present invention can be administeredto an individual via any route known in the art, including, but notlimited to, those disclosed herein. Preferably administration of thepolyamine analog conjugates is intravenous. Other methods ofadministration include but are not limited to, oral, intrarterial,intratumoral, intramuscular, transdermal or transcutaneous,subcutaneous, intraperitoneal, gastrointestinal, and directly to aspecific or affected organ, e.g., the prostate.

The polyamine analog conjugates described herein are administratable inthe form of tablets, pills, powder mixtures, capsules, injectables,solutions, suppositories, emulsions, dispersions, food premixes, and inother suitable forms. Additional methods of administration are known inthe art. The pharmaceutical dosage form which contains the compoundsdescribed herein is conveniently admixed with a non-toxic pharmaceuticalorganic carrier or a non-toxic pharmaceutical inorganic carrier. Typicalpharmaceutically-acceptable carriers include, for example, mannitol,urea, dextrans, lactose, potato and maize starches, magnesium stearate,talc, vegetable oils, polyalkylene glycols, ethyl cellulose,poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropylmyristate, benzyl benzoate, sodium carbonate, gelatin, potassiumcarbonate, silicic acid, and other conventionally employed acceptablecarriers. The pharmaceutical dosage form may also contain non-toxicauxiliary substances such as emulsifying, preserving, or wetting agents,and the like. A suitable carrier is one which does not cause anintolerable side effect, but which allows the conjugates to retain itspharmacological activity in the body. Formulations for parenteral andnonparenteral drug delivery are known in the art and are set forth inRemington 's Pharmaceutical Sciences, 19th Edition, Mack Publishing(1995). Solid forms, such as tablets, capsules and powders, can befabricated using conventional tableting and capsule-filling machinery,which is well known in the art. Solid dosage forms can contain anynumber of additional non-active ingredients known to the art, includingexcipients, lubricants, dessicants, binders, colorants, disintegratingagents, dry flow modifiers, preservatives, and the like.

Liquid forms for ingestion can be formulated using known liquidcarriers, including aqueous and non-aqueous carriers, suspensions,oil-in-water and/or water-in-oil emulsions, and the like. Liquidformulations can also contain any number of additional non-activeingredients, including colorants, fragrance, flavorings, viscositymodifiers, preservatives, stabilizers, and the like. For parenteraladministration, polyamine analog or quinone conjugates can beadministered as injectable dosages of a solution or suspension of thecompound in a physiologically acceptable diluent or sterile liquidcarrier such as water or oil, with or without additional surfactants oradjuvants. An illustrative list of carrier oils would include animal andvegetable oils (peanut oil, soy bean oil), petroleum-derived oils(mineral oil), and synthetic oils. In general, for injectable unitdoses, water, saline, aqueous dextrose and related sugar solutions, andethanol and glycol solutions such as propylene glycol or polyethyleneglycol are preferred liquid carriers. The amount of conjugateadministered per administration will vary, depending on the conditionbeing treated and the individual's medical history.

The pharmaceutical unit dosage chosen is preferably fabricated andadministered to provide a final concentration of drug at the point ofcontact with the cancer cell of from about 1 μM to about 10 mM. Morepreferred is a concentration of from about 1 μM to about 100 μM; stillmore preferred is a concentration of from about 1 μM to about 50 μM. Aswith all pharmaceuticals, the optimal effective concentration of apolyamine analog conjugate or quinone conjugate will need to bedetermined empirically and will depend on the type and severity of thedisease, route of administration, disease progression and health andmass or body area of the patient. Such determinations are within theskill of one in the art. Polyamine analog conjugates or quinoneconjugates can be administered as the sole active ingredient, or can beadministered in combination with another active ingredient, including,but not limited to, cytotoxic agents, antibiotics, antimetabolites,nitrosourea, and vinca alkaloids.

Therapy may be monitored using standard methods in the art, such asdetermination of PSA levels in blood, biopsy, or imaging of the prostateor other tissue or organ.

The following examples are provided to illustrate but not limit theinvention.

EXAMPLES Synthesis of Conformationally-Restricted Polyamine Analogs

a) Spermine and Homospermine Analogs Containing a ConformationalRestriction

Scheme 2 exemplifies a N^(α), N¹⁰⁷ -bisethyl homospermine analog 7containing a central trans-unsaturated bond. Amide 4 was prepared asdescribed in Scheme 1 by alkylation of amide 1 with bromobutyronitrileto give 2, followed by reduction of the nitrile to the amine 3 that wasmesitylsulfonated to 4. Trans-allylic diester 5 was used to alkylateamide 4 and the tetramide 6 was obtained. Deprotection gave thetrans-tetramide 7 (Scheme 2). Introduction of a triple bond in thebutane segment of homospermine also reduces its mobility. This wasachieved by starting with the butyne diester 8 and following thesequence of reactions outlined above (Scheme 3). Schemes 15-20 arefurther examples of the synthesis of polyamine spermine and homospermineanalogs of this type.

b) Synthesis of Pentamines with Conformational Restrictions.

Schemes 4-14 are outlines of the syntheses of conformationallyrestricted pentamines. Scheme 4 depicts the reaction ofcis-1-chloro-4-phthalimido butene with amide 1 to give 11.Hydrazinolysis of 11 gave 12 which was amidated to 13. Reaction of thelatter with 1,4-diiodobutane gave 14, while reaction with equimolaramounts of cis-1,4-dichlorobutene gave 15.

Amide 4 was alkylated with either 4-chlorobutyronitrile to give 16 orwith cis-1,4-dichlorobutene to give 19. Nitrile 16 was reduced withhydrogen over Ni Raney to the amine 17 and the latter transformed in tothe amide 18 (Scheme 5). Condensation of 18 with the chloroalkylintermediate 15 gave the pentamide 20 that was deprotected to thepentamine 21 (Scheme 6). Condensation of 18 with the iodoalkylderivative 14 gave 22 that was deprotected to the pentamine 23 (Scheme7). Condensation of 18 and 19 gave pentamide 24 that was deprotected tothe pentamine 25 (Scheme 8). Using 14 as the alkylating agent,mesitylenesulfonamide was dialkylated to give 26, and the latterdeprotected to give 27 (Scheme 9). The analogous reaction carried outusing 15 as alkylating agent, gave 28 and after deprotection led to thepentamine 29 (Scheme 10).

Alkylation of mesitylenesulfonamide with 19 gave the pentamide 30, whichwas deprotected to 31 (Scheme 11). When 19 was used to alkylate anequimolar amount of mesitylenesulfonamide then 32 was obtained.Alkylation of 32 with 14 gave 33, that was deprotected to give 34(Scheme 12). When the chloroalkyl intermediate 15 was used to alkylateone equivalent of mesitylenesulfonamide, then the triamide 35 wasobtained. Reaction of 35 with 14 gave 36 which was then deprotected to37 (Scheme 13). Condensation of 35 and 19 gave the pentamide 38 that wasdeprotected to 39 (Scheme 14). The above mentioned Schemes describe thesynthesis of cis-compounds. The same synthetic methodology can be usedto obtain the trans-isomers, or cis and trans bonds in differentsegments within the same molecule.

c) Polyamine Analog with Diamidine Substituents.

A new class of polyamine analogs is shown in Scheme 21. They derive from1,4-dibenzylputrescine, 1,5-dibenzylcadaverine, and1,6-dibenzylhexanediamine. They are diamidine derivatives, where thediamidine residues are carrier groups that have been shown to beefficient in the transport of drugs into different protozoa. The generalprocedure of synthessis was based on the condensation of4-cyanobenzaldehyde with the diaminoalkanes to give the Schiff bases,followed by reduction in situ to the corresponding dinitriles 68. Thelatter were converted to the diamidines 69 through their iminoethers.

d) Synthesis of Oligoamines.

Scheme 22 describes the synthesis of a N-2 hydroxyethyl derivative of apentamine such as 75. Starting wtih 18, alkylation with4-bromobutyronitrile gave 70. Reduction of the nitrile of 70 andmesitylenesulfonylation of the resulting amino group gave 71. It wasalkylated again with 4-bromobutyronitrile to give 72, and again reducedand mesitylsulfonylated to give 73. The latter was then alkylated withthe benzyl ester of 2-bromoethanol to give 74. Treatment withhydrobromic acid in acetic acid cleaved both the mesitylene sulfonylprotecting groups and the benzyl ether residue to give 75.

Scheme 23 reports the synthesis of a trans-decamine 77 and of acis-decamine 79. Starting with the pentamide 73 (Scheme 22) and byreaction with trans-diester 5 (Scheme 2) the decamide 76 was prepared,which on deprotection gave 77 as a decahydrochloride. In an analogousmanner, by condensation of 73 with the cis-1,4-dimesityleneoxy-2-butene,the decamide 78 was prepared, which on deprotection gave 79 as adecahydrochloride.

Scheme 24 outlines the synthesis of a N-2 hydroxyethyl trans-decamine 92and a cis-2-hydroxyethyl decamine 95. The procedure repeats almost allthe procedures described in the foregoing schemes. The synthesis of 80proceeded by alkylating BOC-mesitylenesulfonamide with the benzyl esterof 2-bromoethanol. Cleavage of the BOC protecting group leads to 81,alkylation with 4-bromobutyronitrile then gave 82, and after reductionof the nitrile group and reaction with mesitylene sulfonyl chloride thediamide 83 was obtained. Again, alkylation with 4-bromobutyronitrile ledto 84, reduction and mesitylsulfonylation gave 85, alkylation of 85 gave86, reduction and mesitylsulfonylation gave 87, and alkylation,reduction and mesitylsulfonylation performed on 87 gave 89. Alkylationof 73 with trans-1,4-dibromo-2-butene gave 90. Alkylation of 89 with 90gave 91, which after deprotection gave the trans-ω-hydroxy-decamine 92.Alkylation of 73 with cis-1,4-dichloro-2-butene gave 93. Alkylation of89 with 93 gave 94. Deprotection of 94 gave the cis-ω-hydroxy-decamine95, isomeric with 92.

e) Synthesis of Oligoamine Dipeptides.

Scheme 25 outlines the synthesis of a dipeptide derivative of 75(SL-11141) that can be considered as a substrate of cathepsin B.Starting with 74, hydrogenolysis leads to 96, that is then is thenesterified with N-BOC-glutamine to 97. The N-BOC residue is cleaved withTFA and N-BOC-leucine is coupled to the glutanine residue to give 98.Deprotection in acid media then affords the dipeptide 99 (SL-11155).

(f) Synthesis of Polyamine Conjugates of Peptides

Scheme 26 outlines the synthesis of polyamine conjugates of theN-morpholino derivative of tetrapeptide SKLQ, the minimal structuralrequirement of a substrate of PSA. The protected form of the peptide(N-BOC residues) will be conjugated at its carboxy terminal with thepolyamine residues corresponding to SL-11047, SL-11101, or BE-4-4-4-4 togive the conjugates 112, 113, and 114. The polyamine intermediates areconstructed as follows. Chloride 100 is condensed with 46 to give 101.The phthalimido group is cleaved by hydrazynolysis to give 102, and thelatter is mesitylated to 103 This amide is again alkylated with 104 togive 105. The mesitylene sulfonyl groups of 105 are then cleaved and 106is obtained. It is protected using (BOC)₂O, and the resulting 102 isdeprotected by hydrazynolysis to give the polyamine moiety of 112. Intandem, the known 74 (Scheme 17) was alkylated with 108 to give 109.Cleavage of the mesitylenesulfonyl groups gave 110. The free aminogroups were reprotected with (BOC)₂O to give 111. Cleavage of thephthalimido residue via hydrazinolysis using a procedure analogous tothat for compound 12 below gave the aminopolyamine intermediate for thesynthesis of 114.

Should a secondary amine be desired in place of the primary amino groupof compounds 112-114 and analogous compounds, the primary amine can bereadily alkylated under basic conditions with an alkyl halide to yield asecondary amine. As this amine remains unprotected, while the otheramines are still protected by the BOC groups, coupling of the peptide tothe secondary amine can be accomplished using the same protocol as givenabove for the primary amines; the reaction time for the coupling mayneed to be extended and the progress of the reaction can be readilymonitored by HPLC or other methods.

Example 1 Synthesis of Polyamine Compounds

Compound 2: NaH (80%, 1.08 g, 36 mmol) was added to a solution of amide1 (6.81 g, 30 mmol) in DMF (50 ml) in an ice-water bath under N₂. Themixture was stirred for 1 h and a solution of 4-bromobutyronitrile (4.88g, 33 mmol) in DMF (10 ml) was added in portions. The mixture wasstirred over night at 75° C. The solvent was distilled off, the residuetaken up in chloroform washed with a saturated solution of ammoniumchloride, dried (Na₂SO₄) and evaporated. The residue was purifid byflash chromatography on silica gel (hexane/ethyl acetate 3:1) to yield8.0 g (90%) of 2 as a colorless oil. ¹H-NMR (CDCl₃) δ 1.05 (t, 3H), 1.90(m, 2H), 2.30 (b, m, 5H), 2.60 (s, 6H), 3.20 (q, 2H), 3.35 (t, 2H), 6.95(s, 2H); ¹³C-NMR (CDCl₃): δ 12.50, 20.61, 22.43, 23.60, 31.05, 36.12,40.39, 43.78, 118.62, 131.79, 132.67, 139.71, 142.41. MS-EI (m/z) 294(M⁺).

Compound 4: Nitrile 2 (7.8 g, 27 mmol) was dissolved in a mixture ofethanol (150 ml) and concentrated hydrochloric acid (1.5 ml). PtO₂ wasadded (700 mg) and the mixture was hydrogenated at 50 psi over night.The catalyst was filtered off and the solvent evaporated. The residue(78 g, 98%) was used in the next step without further purification. Thefree base gave ¹H-NMR (CDCl₃) δ 1.00 (t, 3H), 1.55 (m, 4H), 2.25 (s,3H), 2.80 (t, 2H), 3.20 (m, 4H), 6.95 (s, 2H); ¹³C-NMR (CDCl₃): δ 12.54,20.69, 22.53, 24.72, 27.65, 39.92, 40.29, 44.59, 131.71, 133.21, 139.82,142.09. FAB-MS (m/z) 299 (M⁺+1). Mesitylenesulfonyl chloride (8.8 g,40.5 mmol) in dioxane (30 ml) was added dropwise to a stirred mixture ofcompound 3 (7.8 g, 27 mmol) dissolved in dioxane (60 ml) and 50% KOH (30ml) at 0° C. The reaction mixture was allowed to reach 20° C. and thenkept over night. An excess of water was added and the mixture wasextracted with chloroform, dried (Na₂SO₄) and evaporated. The oilyresidue was crystallized from ethyl acetate/hexane yielding 4; 10.9 g(82%); mp 71.5-72° C. ¹H-NMR (CDCl₃) δ 1.00 (t, 3H), 1.10-1.50 (m, 4H),2.30 (s, 6H), 2.55, 2.60 (s, 12H), 2.85 (q, 2H), 3.15 (m, 4H), 4.70 (t,1H), 6.95, 7.00 (s, 4H); ¹³C-NMR (CDCl₃): δ 12.70, 20.92, 21.04, 22.73,22.92, 24.58, 26.68, 40.04, 42.02, 44.42, 131.91, 133.31, 133.64,138.99, 140.05, 142.15, 142.35. MS-FAB (m/z) 480 (M⁺).

(E)-2-Butene-1,4-diyl bis[mesitylenesulfonate] (5):(E)-2-Butene-1,4-diol (1.76 g, 20 mmol), and benzyltriethylammoniumbromide (270 mg, 1 mmol) were dissolved in 30 ml of a 50% potassiumhydroxide solution and 30 ml of dioxane. The mixture was stirred at 5°C. and mesitylenesulfonyl chloride (8.72 g, 40 mmol) dissolved in 30 mlof dioxane was added dropwise. When the addition was over, stirring wascontinued for 1 h, water was then added, and the white precipitate wasfiltered and crystallized from chloroform-hexane to yield 5; 7.0 g(77%); mp 119-120° C. ¹H-NMR (CDCl³): δ 2.35 (s, 6H), 2.60 (s, 12H),4.45 (d, 4H), 5.75 (b, 2H), 6.95 (s, 4H); ¹³C-NMR (CDCl₃): δ 20.96,22.52, 67.96, 127.67, 131.69, 131.74, 139.79, 143.45. MS-EI (m/z), 452(M⁺), 253, 200, 183.

Anal. Calcd for C₂₂H₂₈O₆S₂: C, 58.40; H, 6.19. Found: C, 58.35; H, 6.22.

Compound 6 was synthesized from 5 according to a procedure describedelsewhere (Reddy et al., J. Med. Chem. 41:4723 (1998)) in 56% yield.¹H-NMR (CDCl₃): δ 0.95 (t, J=7.15 Hz, 6H, CH₃), 1.34 (m, 8H, CH₂), 2.29(s, 12H, CH₃), 2.55 (s, 24H, CH₃), 3.09 (m, 12H, NCH₂), 3.72 (d, J=4.53Hz, 4H, NCH₂), 5.48 (t, J=4.31 Hz, 2H, CH═CH), 6.92 (s, 4H, Ph), 6.93(s, 4H, Ph); ¹³C-NMR (CDCl₃): δ 12.71, 20.90, 22.71, 22.76, 24.74,40.04, 42.21, 44.56, 45.69, 128.45, 131.88, 132.02, 140.05, 140.16,142.20, 142.58. MS-FAB (m/z) 1012.8 (M⁺, 100%), 828.7, 646.7, 561, 176.

Compound 7 was obtained from 6 as described elsewhere (Reddy et al., J.Med. Chem. 41:4723 (1998)) in 75% yield, mp>230° C. ¹H-NMR (D₂O): δ 1.26(t, J=12.5 Hz, 6H, 2CH₃), 1.79 (m, 8H, CH₂), 3.12 (m, 12H, NCH₂), 3.80(d, J=7.16, 4H, NCH₂), 6.10 (m, 2H, CH═CH); ¹³C-NMR (D₂O): δ 12.79,25.10, 45.19, 48.53, 48.62, 50.36, 130.66. MS-MALDI (m/z): 285.3 (MH⁺,100%).

Compound 8 was obtained from the commercially available butyne diol.Mesitylenesulfonyl chloride (19.5 g, 90 mmol) in dioxane (30 ml) wasadded dropwise to a stirred and cooled mixture of butyne diol (2.58 g,30 mmol), 50% potassium hydroxide (30 ml) and triethylbenzyne ammoniumbromide (405 mg, 1.5 mmol). Once the addition was over, the mixture wasstirred at room temperature for an additional 3 h. An excess of waterwas added and the white precipitate was cooled over night, filtered offand dried. Recrystallization from ethyl acetate/hexane afforded 8.6 g(63%) of 8; mp 105-106° C. ¹H-NMR (CDCl₃): δ 2.30 (s, 6H), 2.60 (s,12H), 4.50 (s, 4H), 6.95 (s, 4H); ¹³C-NMR (CDCl3): δ 20.93, 22.48,56.13, 80.41, 130.65, 131.67, 139.98, 143.67. MS-EI (m/z) 450 (M⁺).

Compound 9 was obtained following a procedure analogous to thatdescribed for compound 42 (see below). From 450 mg (1 mmol) of diester 8and 1.05 g (2.2 mmol) of diamide 4, 570 mg (56%) of tetramide 9 wasobtained. ¹H-NMR (CDCl₃): δ 0.90 (t, 6H), 1.30 (bs, 8H), 2.20 (s, 12H),2.45 (s, 24H), 3.05 (m, 12H), 3.75 (s, 4H), 6.87 (s, 8H); ¹³C-NMR(CDCl₃): δ 12.70, 20.78, 22.68, 34.65, 39.97, 44.46, 44.99, 78.62,131.85, 131.98, 132.34, 140.14, 142.13, 142.55. MS-FAB (m/z) 1010(M^(⊕)).

Compound 10 was obtained following a procedure analogous to thatdescribed for compound 43 (see below). From 500 mg (0.49 mmol) oftetramide 9, 160 mg (76%) of the tetrahydrochloride 25 was obtained;mp>280° C. (decomp). ¹H-NMR (D₂O): δ 1.30 (t, 6H), 1.80 (b, 8H),2.90-3.25 (m, 12H), 4.05 (s, 4H); ¹³C-NMR (D₂O): δ 13.39, 25.64, 39.26,45.72, 49.00, 49.20, 81.20. MS-MALDI 283 (M⁺+1).

Compound 11: Mesitylenesulfonylethylamide 1 (3.1 g, 13.65 mmol) wasdissolved in anhydrous DMF (30 ml) followed by the addition of NaH (85%,0.423 g) in several portions. The mixture was stirred at roomtemperature for 1 h. N-(4-chloro-2-butenyl)-phthalimide (Aldrich, 3.06g, 13 mmol) in 20 ml of DMF was added to the flask followed by stirringat 80° C. over night. The mixture was cooled to room temperature,quenched with H₂O (10 ml), and the solution was evaporated to dryness invacuo. The solid residue was partitioned between 25 ml H₂O and 25 CHCl₃.The aqueous layer was extracted with CHCl₃ (3×25 ml), the organic layerswere washed with brine (35 ml), dried (MgSO₄), the solvent wasevaporated to afford a gum which solidified upon trituration with hexaneto give 11. The ¹H-NMR and ¹³C-NMR spectra showed that 11 was pureenough to be used in the next step without further purification, yield4.75 g. ¹H-NMR (CDCl₃): δ 1.16 (t, J=7.11 Hz, 3H, CH₃), 2.29 (s, 3H,CH₃), 2.63 (s, 6H, 2CH₃), 3.29 (q, J=7.11 Hz, 2H, CH₂), 4.06 (d, J=5.24Hz, 2H, NCH₂), 4.26 (d, J=5.72 Hz, 2H, NCH₂), 5.59 (m, 2H, CH═CH), 6.95(s, 2H, Ph), 7.71 (m, 2H, Ph), 7.83 (m, 2H, Ph); ¹³C-NMR (CDCl₃): δ13.06, 20.89, 22.72, 34.35, 40.68, 42.01, 123.27, 126.69, 129.47,131.90, 134.00, 140.24.

Compound 12: Amide 11 (20 g, 46.95 mmol) was dissolved in methanol,hydrazine monohydrate (5 ml, 98.52 mmol) was added and the solutionstirred at 55° C. for 24 h. Initially it was a homogeneous solution;however, after several hours a white solid precipitated. The mixture wascooled to room temperature, 300 ml of conc. HCl were added slowly(exothermic reaction), and stirring at room temperature was continuedfor 12 h more. Methanol was evaporated, and the resulting solid wasextracted with CHCl₃ (3×150 ml). The aqueous layer was neutralized with50% NaOH, extracted again with CHCl₃ (3×100 ml), the combined organiclayers were dried (MgSO₄); the solution was evaporated to afford a gum,which solidified under high vacuum to give 12; yield 9.0 g (65%). Thecompound was purified by column chromatography using hexane, ethylacetate (7:3) as eluent; mp 167-169° C. ¹H-NMR (CDCl₃): δ 1.0 (t, J=7.1Hz, 3H, CH₃), 2.28 (s, 3H, CH₃), 2.56 (s, 6H, 2CH₃), 2.62 (br, NH₂),3.12 (q, J=7.1 Hz, 2H, NCH₂), 3.73 (br, 2H, NCH₂), 3.94 (d, J=6.0 Hz,2H, NCH₂), 5.80 (m, 2H, CH═CH), 6.92 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ12.97, 20.93, 22.74, 36.43, 40.94, 42.08, 124.29, 131.89, 132.00,132.62, 140.21, 142.67.

Compound 13 was obtained from 12 as described for 4 in 96% yield. It waspurified by column chromatography using hexane and ethyl acetate (4:1.5)as eluants; mp 98-99° C.; ¹H-NMR (CDCl₃): δ 0.93 (t, J=5.85 Hz, 3H,CH₃), 2.23 (s, 3H, CH₃), 2.24 (s, 3H, CH₃), 2.50 (s, 6H, 2CH₃), 2.56 (s,6H, 2CH₃), 3.06 (q, J=7.15 Hz, 2H, NCH₂), 3.48 (t, J=5.99 Hz, 2H, NCH₂),3.68 (d, J=5.72 Hz, 2H, NCH₂), 4.58 (t, J=6.24 Hz, 1H, NH), 5.44 (m, 2H,CH═CH), 6.87 (s, 2H, Ph), 6.89 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ 12.80,20.89, 22.64, 22.89, 39.01, 40.59, 41.41, 128.14, 128.46, 131.91,131.96, 139.08, 140.19, 142.26, 142.54. MS-FAB (m/z) 479.2 (M⁺, 65%),296.2, 279.1, 267.2, 183.1.

Compound 15: Amide 13 (4.79 g, 10 mmol) was dissolved in anhydrous DMF(40 ml) followed by addition of NaH (0.37 g) in several portions, themixture stirred at room temperature for 2 h, cis-1,4-dichloro-2-butene(7.5 g, 60 mmol) in 10 ml DMF was added at once, and stirring wascontinued at 50° C. over night. The mixture was cooled to roomtemperature, quenched with 10 ml H₂O, the solvents were evaporated, andthe contents were partitioned between H₂O (50 ml) and CHCl₃ (50 ml). Theaqueous layer was extracted with CHCl₃ (3×50 ml), the pooled organiclayers were dried (MgSO₄), evaporated, and 15 was purified by columnchromatography using hexane, ethyl acetate (8.5:1.5) as eluants; yield5.5 g (97%), mp 106-108° C. ¹H-NMR (CDCl₃): δ 1.03 (t, J=7.33 Hz, 3H,CH₃), 2.30 (s, 6H, 2CH₃), 2.57 (s, 12H, 4CH₃), 3.17 (q, J=7.31 Hz,NCH₂), 3.71 (m, 4H, NCH₂), 3.81 (d, J=6.87 Hz, 2H, NCH₂), 3.95 (d,J=7.70 Hz, 2H, CHCl₂), 5.50 (m, 3H, CH═CH), 5.74 (m, 1H, CH═CH), 6.93(s, 2H, Ph), 6.95 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ 12.91, 22.70, 22.74,38.20, 40.45, 41.60, 42.11, 42.33, 128.17, 128.95, 129.34, 129.40,131.94, 132.08, 140.23, 140.34, 142.91. MS-FAB (m/z) 566.7 (M⁺, 100%),153.4, 96.3.

Compound 14 was prepared from 13 and 1,4-diiodobutane as described abovefor 15. The product was purified by column chromatography using hexanesand ethyl acetate (4:1) as eluant; yield 79%. ¹H-NMR (CDCl₃): δ 1.04 (t,J=7.10 Hz, 3H, CH₃), 1.63 (m, 4H, CH₂), 2.30 (s, 6H, 2CH₃), 2.58 (s,12H, 4CH₃), 3.04 (t, J=6.50 Hz, 2H, CH₂I), 3.16 (m, 4H, NCH₂), 3.78 (d,J=5.14 Hz, 4H, NCH₂), 5.55 (m, 2H, CH═CH), 6.94 (s, 2H, Ph), 6.95 (s,2H, Ph); ¹³C-NMR (CDCl₃): δ 5.69, 12.92, 20.95, 22.72, 22.78, 28.25,30.36, 40.47, 41.59, 42.11, 44.71, 128.34, 129.00, 131.94, 132.06,132.60, 132.89, 140.15, 140.21, 142.50, 142.71.

Compound 16 was prepared from 4 and 4-bromobutyronitrile as describedabove for Compound 2 in 94% yield.

¹H NMR(CDCl₃): δ 0.97 (t, J=7.12 Hz, 3H, CH₃), 1.40 (m, 4H, 2CH₂), 1.85(Pent., m, 2H, CH₂), 2.27 (t, J=7.17 Hz, 2H CH₂CN), 2.30 (s, 6H, 2CH₃),2.57 (s, 6H, 2CH₃), 2.58 (s, 6H, 2CH₃), 3.13 (m, 6H, NCH₂), 3.28 (t,J=7.11 Hz, 2H, NCH₂), 6.94 (s, 2H, Ph), 6.96 (s, 2H, Ph); ¹³C NMR(CDCl₃): δ 12.55, 14.54, 20.84, 22.64, 22.73, 23.65, 24.43, 24.57,39.88, 44.31, 44.54, 45.58, 118.69, 131.84, 132.05, 132.73, 133.36,139.94, 142.20, 142.71.

Compound 17 was prepared from 16 as described above for Compound 3 in93% yield. ¹H NMR(CDCl₃): δ 1.00 (t, J=6.92 Hz, 3H, CH₃), 1.40 (m, 10H,4CH₂, NH₂), 2.29 (s, 6H, 2CH₃), 2.57 (b, 14H, 4CH₃, CH₂N), 3.13 (m, 8H,4CH₂N), 6.93 (s, 4H, 2 Ph); ¹³C NMR (CDCl₃): 12.72, 20.90, 22.72, 22.78,24.67, 24.80, 30.80, 40.02, 41.61, 44.56, 45.10, 45.38, 131.87, 140.04,142.21, 142.28; MS-FAB(M/Z) 552.3(M⁺, 100%), 368.2, 299.1, 183.0, 154.0.

Compound 18 was prepared from 17 as described above for Compound 4.

¹H NMR(CDCl₃): δ 0.96 (t, J=7.13 Hz, 3H, CH₃), 1.38 (m, 8H, 4CH₂), 2.29(s, 9H, 3CH₃), 2.55 (s, 6H, 2CH₃), 2.56 (s, 6H, 2CH₃); 2.59 (s, 6H,2CH₃), 2.80 (m, 2H, CH₂N), 3.10 (m, 8H, NCH₂), 4.67(t, J=6.6 Hz, 1H,NH), 6.93 (s, 6H, 3 Ph); ¹³C NMR(CDCl₃): δ 12.56, 20.87, 22.70, 22.74,22.84, 24.40, 26.45, 24.67, 26.62, 39.87, 41.88, 44.45, 45.02, 45.09,131.86, 131.90, 131.92, 133.12, 133.32, 133.68, 138.91, 139.97, 142.02,142.21, 142.38; MS-FAB(M/Z): 756.9(M+23(Na), 100%) 572.8, 390.7, 333.6,305.6

Compound 19 was prepared from 4 and 1,4-dichloro-2-butene as describedabove for 15 in 99% yield. ¹H-NMR (CDCl₃): δ 1.01 (t, J=7.11 Hz, 3H,CH₃), 1.38 (m, 4H, CH₂), 2.29 (s,3H), 2.30 (s,3H), 2.57 (s, 6H), 2.61(s, 6H), 3.11 (m, 4H, NCH₂), 3.16 (q, J=7.15 Hz, 2H, NCH₂), 3.81 (d,J=7.17 Hz, 2H, NCH₂), 3.98 (d, J=8.05 Hz, 2H, CH₂Cl), 5.51 (m, 1H,CH═CH), 5.77 (m, 1H, CH—CH), 6.93 (s, 2H, Ph), 6.95 (s, 2H, Ph); ¹³C-NMR(CDCl₃): δ 12.76, 20.91, 22.71, 22.76, 24.74, 38.12, 40.08, 41.85,44.59, 45.54, 129.14, 129.25, 131.88, 132.02, 140.09, 140.19, 142.21,142.63. MS-FAB (m/z) 569.3 (M⁺, 20%), 385.2, 240.1, 203.3, 183.0, 119(100%).

Compound 20 was prepared from 18 and 15 following the proceduredescribed above for 15. It was purified by column chromatography usinghexanes-ethyl acetate (7:3) as eluant (78% yield). ¹H-NMR (CDCl₃): δ0.97 (t, J=7.10 Hz, 3H, CH₃), 0.99 (t, J=7.0 Hz, 3H, CH₃), 1.29 (m, 8H,CH₂), 2.29 (s, 15H, CH₃), 2.54, 2.55, 2.59 (s, 30H, CH₃), 3.06 (m, 12H,NCH₂), 3.65 (m, 8H, NCH₂), 5.48 (m, 4H, CH═CH), 6.92 (s, 10H, Ph);¹³C-NMR (CDCl₃): δ 12.70, 12.83, 20.88, 20.91, 22.65, 22.68, 22.72,22.74, 24.48, 24.72, 40.04, 40.47, 41.53, 42.07, 42.22, 42.34, 44.54,44.96, 127.94, 128.27, 128.57, 129.20, 131.92, 132.05, 139.96, 140.00,140.12, 140.16, 140.27, 142.19, 142.25, 142.47, 142.58, 142.87. MS-FAB(m/z) 1263.81 (M⁺, 100%), 1080.01, 898.11, 714.81, 563.

Compound 21: Pentamide 20 (0.93 g, 0.735 mmol) was dissolved in 20 mlanhydrous CH₂Cl₂, phenol (3.46 g, 36.77 mmol) was added, followed by HBrin acetic acid (30%, 17.62 ml) and the mixture was stirred over night at25° C. Water (10 ml) was added to the flask, the aqueous layer wasseparated, the organic layer was extracted with 5 ml H₂O, and thecombined aqueous layers were washed with CH₂Cl₂ (6×15 ml). Water wasevaporated under vacuum to afford a solid which was dissolved in 1 ml 1NNaOH followed by 1 ml of 50% KOH. This solution was extracted with CHCl₃(10×5 ml). The combined organic layers were dried (MgSO₄), CHCl₃ wasevaporated, and the residue dissolved in anhydrous diethyl ether.Anhydrous HCl gas was passed into the solution while cooling at 0° C. Awhite solid precipitated which was filtered and washed with ether. Itwas 21 (84%). ¹H-NMR (D₂O): δ 1.29 (t, J=7.32 Hz, 3H, CH₃), 1.31 (t,J=7.24 Hz, 3H, CH₃), 1.79 (m, 8H, CH₂), 3.12 (m, 12H, NCH₂), 3.87 (m,8H, NCH₂), 5.98 (m, 4H, CH═CH); ¹³C-NMR (D₂O): δ 13.36, 13.46, 25.66,25.77, 45.44, 45.74, 46.24, 46.41, 46.84, 49.09, 49.41, 49.70, 129.02,129.16, 129.47, 129.66. MS-MALDI (m/z) 354.36 (MH⁺, 100%).

Compound 22 was prepared in 51% yield from 18 and 14 as described abovefor compound 15. ¹H-NMR (CDCl₃): δ 0.97 (t, J=6.59 H, 3H, CH₃), 0.99 (t,J=7.02 Hz, 3H, CH₃), 1.29 (m, 12H, CH₂), 2.29 (s, 15H, CH₃), 2.55 (s),2.56 (s), 2.57 (s), 3.10 (m, 16H, NCH₂), 3.70 (m, 4H, NCH₂), 5.47 (m,2H, CH═CH), 6.93 (s, 10H, Ph); ¹³C-NMR (CDCl₃): δ 12.69, 12.83, 20.91,22.69, 22.71, 22.76, 24.43, 24.70, 40.48, 41.11, 41.48, 44.50, 44.91,128.13, 128.90, 131.88, 131.94, 132.01, 133.29, 139.95, 140.00, 140.15,142.22, 142.29, 142.60. MS-FAB (m/z) 1265.91 (M⁺, 100%), 1082.01,900.11, 716.91, 563.81.

Compound 23 was prepared from 22 in 79% yield as described above for 21.¹H-NMR (D₂O): δ 1.29 (t, J=7.29 Hz, 3H, CH₃), 1.30 (t, J=7.30 Hz, 3H,CH₃), 1.78 (m, 12H, CH₂), 3.12 (m, 16H, NCH₂), 3.83 (m, 4H, NCH₂), 5.96(m, 2H, CH═CH); ¹³C-NMR (D₂O): δ 13.31, 13.42, 25.62, 25.75, 45.38,45.71, 46.18, 46.76, 49.07, 49.32, 49.69, 129.11, 129.39. MS-MALDI (m/z)356.38 (MH⁺, 100%).

Compound 24 was prepared from 18 (52% yield) as described. ¹H-NMR(CDCl₃): δ 0.95 (m, 6H, 2CH₃), 1.32 (m, 12H, CH₂), 2.29 (s, 15H, CH₃),2.55 (s, 30H, CH₃), 3.06 (m, 16H, NCH₂), 3.70 (m, 4H, NCH₂), 5.47 (m,2H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR (CDCl₃): δ 12.67, 20.90, 22.71,22.76, 24.43, 24.68, 39.97, 42.08, 44.48, 44.90, 45.61, 128.28, 128.45,131.87, 131.93, 132.01, 139.96, 140.00, 140.12, 142.21, 142.28, 142.58.MS-FAB (m/z) 1265.91 (M⁺, 100%), 1082.01, 900.11.

Compound 25 was prepared from 24 in 96% yield as described above for 21.¹H-NMR (D₂O): δ 1.28 (t, J-7.29 Hz, 6H, 2CH₃), 1.78 (m, 12H, CH₂), 3.09(m, 16H, NCH₂), 3.84 (m, 4H, NCH₂), 5.96 (m, 2H, CH═CH); ¹³C-NMR (D₂O):δ 13.31, 25.61, 25.73, 45.70, 46.79, 49.05, 49.36, 49.65, 129.19.MS-MALDI (m/z) 356.4 (MH⁺).

Compound 26: A mixture of KOH (0.25 g), K₂CO₃ (0.25 g) andtetra-n-butyl-ammonium hydrogen bromide (0.05 g) were suspended in 15 mlbenzene. Mesitylenesulfonylamide (0.199 g, 1 mmol) was added to thesuspension and the mixture was heated to 50° C. Iodide 14 (1.98 g, 3mmol) in 10 ml benzene was added to the flask, the mixture heated underreflux over night, then cooled to room temperature; the inorganic solidswere filtered off and washed with benzene (2×20 ml). The combinedorganic layers were washed several times with water until the washingswere neutral. The benzene was dried (MgSO₄), evaporated and the residuepurified by column chromatography using hexanes and ethyl acetate(7.5:2.5) as eluant; 25% yield (0.948 g). ¹H-NMR (CDCl₃): δ 1.00(t,J=7.18 Hz, 6H, CH₃), 1.28 (m, 8H, CH₂), 2.29 (s, 15H, CH₃), 2.53 (s),2.55 (s), 2.57 (s), 3.03 (m, 8H, NCH₂), 3.12 (q, J=7.13 Hz, 4H, NCH₂),3.70 (m, 8H, NCH₂), 5.47 (m, 4H, CH═CH), 6.93 (s, 10H, Ph); ¹³C-NMR(CDCl₃): δ 12.78, 20.85, 22.63, 22.69, 24.32, 24.58, 40.41, 41.43,42.00, 44.76, 45.43, 128.08, 128.83, 131.88, 131.95, 132.77, 132.85,133.23, 139.90, 140.04, 140.08, 142.22, 142.43, 142.53. MS-FAB (m/z)1263.81 (M⁺, 100%), 1081, 898.11, 815.01, 561.81, 418.81.

Compound 27 was prepared from 26 in 57% yield as described above for 21.¹H-NMR (D₂O): δ 1.31 (t, J=7.31 Hz, 6H, CH₃), 1.78 (m, 8H, CH₂), 3.15(m, 12H, NCH₂), 3.83 (m, 8H, NCH₂), 5.96 (m, 4H, CH═CH); ¹³C-NMR(CDCl₃): δ 13.43, 25.64, 25.76, 45.39, 46.19, 46.77, 49.35, 49.72,129.11, 129.41. MS-MALDI (m/z) 354.3 (MH⁺, 100%).

Compound 28 was prepared from 15 and mesitylenesulfonylamide in 24%yield as described above for 26; mp 57.7° C. ¹H-NMR (CDCl₃): δ 0.99 (t,J=7.09 Hz, 6H, CH₃), 2.29 (s, 15H, CH₃), 2.53 (s), 2.55 (s), 3.12 (q,J=7.09 Hz, 4H, NCH₂), 3.63 (m, 16H, NCH₂), 5.49 (m, 8H, CH═CH), 6.93 (s,10H, Ph); ¹³C-NMR (CDCl₃): δ 12.85, 20.89, 20.92, 22.66, 40.47, 41.53,42.19, 128.00, 128.47, 128.58, 129.11, 131.92, 132.05, 140.17, 140.30,142.46, 142.87. MS-FAB (m/z) 1259.81 (M⁺, 60%),1075.91, 894.01, 306.51,153.4 (100%).

Compound 29 was prepared from 28 in 81% yield as described above for 21.¹H-NMR (D₂O): δ 1.31 (t, J=7.29 Hz, 6H, CH₃), 3.15 (q, J=7.31 Hz, 4H,NCH₂), 3.84 (m, 4H, NCH₂), 3.90 (m, 12H, NCH₂), 5.98 (m, 8H, CH═CH);¹³C-NMR (D₂O): δ 13.42, 45.41, 46.22, 46.44, 129.07, 129.37, 129.42,129.58. MS-MALDI (m/z) 350.31 (MH⁺).

Compound 30 was prepared from 19 in 25% yield as described above for 26;mp 62.3° C. ¹H-NMR (CDCl₃): δ 0.95 (5, J=7.17 Hz, 6H, CH₃), 1.33 (m, 8H,CH₂), 2.29 (s, 15H, CH₃), 2.54 (s), 2.55 (s), 3.07 (m, 12H, NCH₂), 3.65(m, 8H, NCH₂), 5.48 (m, 4H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR (CDCl₃):δ 12.69, 20.90, 22.69, 22.73, 24.70, 40.03, 42.13, 42.30, 44.53, 45.59,128.11, 128.79, 131.87, 132.00, 140.02, 140.14, 140.28, 142.17, 142.58,142.85. MS-FAB (m/z) 1263.81 (M⁺, 100%), 1080.01, 898.11, 714.01, 153.

Compound 31 was prepared from 30 in 87% yield as described above for 21.¹H-NMR (D₂O): δ 1.28 (t, J=7.32 Hz, 6H, CH₃), 1.79 (m, 8H, CH₂), 3.10(m, 12H, NCH₂), 3.87 (m, 8H, NCH₂), 5.98 (m, 4H, CH═CH), ¹³C-NMR (D₂O):δ 12.70, 25.00, 25.13, 45.10, 45.81, 46.21, 48.44, 48.78, 128.44,128.85. MS-MALDI (m/z) 354.3 (MH⁺).

Compound 32: Mesitylenesulfonylamide (1.47 g, 7.38 mmol) was dissolvedin 50 ml anhydrous DMF, and NaH (85%, 0.3 g) was added to it under anitrogen atmosphere. The mixture was stirred at room temperature and 19(1.40 g, 2.46 mmol) in 25 ml DMF were added. Heating at 65° C. continuedover night. The mixture was cooled to room temperature, and 10 ml of H₂Owere added. The solvents were evaporated and the solid residue waspartitioned between 40 ml H₂O and 40 ml CHCl₃. The aqueous layer wasextracted with CHCl₃ (2×30 ml), the combined organic layers were washedwith H₂O (3×50 ml), dried (MgSO₄), and evaporated. The residue waspurified by column chromatography using hexanes-ethyl acetate (7.5:2.5).1.7 g (97%) of 32 as a white solid was obtained. ¹H-NMR (CDCl₃): δ 0.94(t, J=7.10 Hz, 3H, CH₃), 1.30 (m, 4H, CH₂), 2.29 (s), 2.30 (s), 2.55 (s,12H, CH₃), 2.65 (s, 6H, CH₃), 3.11 (m, 6H, NCH₂), 3.52 (m, 1H, NCH),3.65 (m, 2H, NCH₂), 3.71 (m, 1H, NCH₂), 4.82 (br, 1H, NH), 5.47 (m, 2H,CH═CH), 6.93 (s, 4H, Ph), 6.96 (s, 2H, Ph); ¹³C-NMR (CDCl₃): δ 12.50,20.91, 22.71, 22.76, 22.83, 22.91, 24.66, 38.98, 39.85, 42.15, 42.26,44.50, 128.06, 128.51, 131.86, 131.91, 138.18, 140.00, 140.14, 140.28,142.17, 142.65.

Compound 33 was prepared from 32 and 14 in 51% yield as described abovefor 22. ¹H-NMR (CDCl₃): δ 0.99 (5, J=7.19 Hz, 6H, CH₃), 1.33 (m, 8H,CH₂), 2.29 (s, 15H, CH₃), 2.55 (s), 2.57 (s), 3.10 (m, 12H, NCH₂), 3.70(m, 4H, NCH₂), 3.77 (m, 4H, NCH₂), 5.42 (m, 4H, CH═CH), 6.93 (s, 10H,Ph); ¹³C-NMR (CDCl₃): δ 12.70, 12.71, 20.89, 22.66, 22.72, 22.78, 22.81,24.60, 26.53, 40.39, 41.37, 41.87, 42.20, 45.47, 128.26, 128.62, 131.78,131.84, 131.86, 131.92, 132.77, 138.92, 139.96, 140.09, 140.17, 142.57,142.63.

Compound 34 was prepared from 33 as described above for 21 in 40% yield.

Compound 35 was prepared from 15 in 94% yield as described above for 32.

Compound 36 was prepared from 35 and 14 in 82% yield as described abovefor 33. ¹H-NMR (CDCl₃): δ 0.99 (t, J=7.11 Hz, 6H, CH₃), 1.33 (m, 4H,CH₂), 2.29 (s, 15H, CH₃), 2.55 (s), 2.57 (s), 3.07 (m, 8H, NCH₂), 3.70(m, 12H, NCH₂), 5.46 (m, 6H, CH═CH), 6.92 (s, 10H, Ph); ¹³C-NMR (CDCl₃):δ 12.69, 12.80, 20.84, 22.62, 22.68, 22.73, 22.77, 24.58, 26.55, 40.44,41.51, 41.86, 42.04, 42.24, 45.49, 128.10, 128.25, 128.52, 128.62,128.82, 131.89, 131.95, 132.79, 138.89, 140.07, 140.14, 140.23, 141.94,142.44, 142.53, 142.82. MS-FAB (m/z) 1262.8 (M⁺, 75%), 1080.01, 896, 119(100%).

Compound 37 was prepared from 36 in 65% yield as described above for 21.¹H-NMR (D₂O): δ 1.31 (t, J=6.97 Hz, 6H, CH₃), 1.79 (m, 4H, CH₂), 3.12(m, 8H, NCH₂), 3.83 (m, 12H, NCH₂), 5.96 (m, 6H, CH═CH); ¹³C-NMR (D₂O):δ 13.48, 25.69, 26.76, 41.67, 45.44, 46.24, 46.45, 46.80, 49.41, 129.00,129.12, 129.45, 129.71. MS-MALDI (m/z) 352.3 (MH⁺).

Compound 38 was prepared from 35 and 19 in 89% yield as described.¹H-NMR (CDCl₃): δ 0.95 (m, 6H, CH₃), 1.33 (m, 4H, CH₂), 2.29 (s, 15H,CH₃), 2.54 (s), 2.55 (s), 2.57 (s), 3.09 (m, 8H, NCH₂), 3.66 (m, 12H,NCH₂), 5.48 (m, 6H, CH═CH), 6.93 (s, 10H, Ph); ¹³C-NMR (CDCl₃): δ 12.51,12.63, 20.84, 20.86, 22.63, 22.65, 22.84, 24.61, 38.92, 40.40, 41.40,42.11, 42.18, 44.44, 45.48, 127.95, 128.07, 128.49, 128.62, 128.80,131.76, 131.83, 131.85, 131.88, 132.01, 138.05, 139.01, 140.07, 140.13,140.24, 142.15, 142.21, 142.87. MS-FAB (m/z) 1263.1 (M⁺, 90%), 1080.1,896.01, 119 (100%).

Compound 39 was prepared from 38 in 54% yield as described above for 21;mp 270° C. (dec.). ¹H-NMR (D₂O): δ 1.31 (m, 6H, CH₃), 1.80 (m, 4H, CH₂),3.10 (m, 8H, NCH₂), 3.86 (m, 12H, NCH₂), 5.98 (m, 6H, CH═CH); ¹³C-NMR(D₂O): δ 13.30, 13.42, 25.58, 25.70, 45.69, 46.21, 46.43, 46.81, 49.02,49.37, 129.00, 129.15, 129.37, 129.59. MS-MALDI (m/z): 352.343 (MH⁺).

Compound 42: NaH (80%, 132 mg, 4.4 mmol) was added to a solution ofdiamide 41 (1.98 g, 4.4 mmol) in DMF (10 ml). The mixture was stirred at20° C. for 30 minutes and a solution of the diester 40 (Reddy et al.(1998) J. Med Chem., 41:4723) (960 mg, 2 mmol) in DMF (10 ml) was addeddropwise. The mixture was stirred at 75° C. for 2 h, the solvent wasdistilled off, the residue was taken in chloroform, washed with asaturated solution of ammonium chloride, dried (Na₂SO₄) and evaporatedto dryness. The crude oil was purified by column chromatography usinghexane-ethyl acetate (8:2) as running solvent. 1.4 g (70%) was obtainedas a glassy oil. ¹³C-NMR (CDCl₃): δ 20.58, 22.63, 22.80, 32.42, 33.86,43.16, 45.42, 46.26, 132.75, 133.21, 139.82, 142.40. MS-FAB 984 (M⁺),

Compound 43: Phenol (1.86 g, 19.7 mmol) and 30% HBr in glacial aceticacid (35 ml) were added in that order to a solution of 42 (600 mg, 0.6mmol) in CH₂Cl₂ (35 ml) at room temperature. The solution was stirredfor 24 h, water (30 ml) was added, followed by extraction with methylenechloride (3×20 ml). The aqueous layer was evaporated under reducedpressure and the residue was taken up in 2N NaOH (2 ml) and then 50% KOH(2 ml) followed by extraction with chloroform (6×10 ml). After removalof chloroform, the residue was taken up in ethanol (15 ml) and acidifiedwith concentrated hydrochloric acid (0.4 ml). The product 43 (230 mg,93%) was recrystallized from aqueous ethanol; mp>270° C. (decomp).¹H-NMR (D₂O): δ 1.95 (m, 2H), 2.05-2.25 (m, 6H), 2.75 (s, 6H), 2.90 (b,2H), 3.10-3.35 (m, 12H); ¹³C-NMR (D₂O): 25.21, 25.24, 35.60, 35.64,47.41, 48.58, 50.87. MS-MALDI (m/z) 240 (M⁺+1).

Compound 47: NaH (80%, 132 mg, 4.4 mmol) was added to a solution ofdiamide 46 (1.98 g, 4.4 mmol) in DMF (10 ml). The mixture was stirred at20° C. for 30 min and a solution of the diester 8 (900 mg, 2 mmol) inDMF (10 ml) was added dropwise. The mixture was stirred at 75° C. for 2h. The solvent was distilled off, the residue was taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(NaSO₄) and evaporated to dryness. The oily residue was crystallizedfrom ethyl acetate/hexane 1.2 g (61%); mp 165-166° C. ¹H-NMR (CDCl₃): δ1.08 (t, 3H), 1.75 (m 4H), 2.28 (s, 12H), 2.55 (bs, 24H), 3.10 (m, 12H),3.98 (s, 4H), 6.95 (m, 8H); ¹³C-NMR (CDCl₃): δ 12.70, 20.86, 22.64,25.14, 34.85, 40.22, 42.62, 43.37, 78.80, 131.99, 132.26, 133.21,140.26, 142.28, 142.71. MS-FAB (m/z) 982 (M⁺).

Compound 48 was obtained as described for 47. From 1.2 g (1.22 mmol) oftetramide 47, 420 mg (86%) of the tetrahydrochloride 48 was obtained;mp>270° C. (decomp). ¹H-NMR (D₂O): δ 1.29 (t, 6H), 2.13 (m, 4H), 3.14(m, 12H), 4.06 (s, 4H); ¹³C-NMR (D₂O): δ 13.34, 25.52, 39.45, 45.90,45.64, 46.71, 81.32. MS-MALDI (m/z) 255 (M⁺+1).

Compound 44 was obtained as described for 47. From 450 mg (1 mmol) ofdiester 8 and 994 mg (2.2 mmol) of diamide 41, 500 mg (52%) of thetetramide 44 was obtained and crystallized from ethyl acetate-hexane; mp155-156° C.

Compound 45 was obtained as described for 43. From 500 mg (0.52 mmol) oftetramide 44, 160 mg (82%) of the tetrahydrochloride 45 was obtained;mp>270° C. (decomp). ¹H-NMR (D₂O): δ 2.15 (m, 4H), 2.73 (s, 3H),3.05-3.40 (m, 8H), 4.10 (s, 4H); ¹³C-NMR (D₂O): δ 25.59, 35.66, 45.90,46.57, 48.61.

Compound 51 is a mixture of cis/trans-isomers. ¹H-NMR (D₂O): δ 1.15-2.10(m, 7H), 2.90 (q, 1H), 3.30-3.80 (b, 2H); ¹³C-NMR (D₂O): δ 24.16, 24.97,28.44, 30.42, 36.58, 37.14, 48.24, 52.27, 55.19, 57.45, 64.55, 67.26.

Compound 52: Mesitylenesulfonylchloride (6.5 g, 30 mmol) in dioxane (10ml) was added dropwise to a stirred and cooled mixture of amine alcohol51 (1.15 g, 10 mmol), triethylbenzyl ammonium bromide (135 mg, 0.5mmol), 50% KOH (10 ml) and dioxane (10 ml). The reaction mixture wasleft over night at 20 ° C. with magnetic stirring. An excess of waterwas added, the solution was extracted with chloroform (3×30 ml), dried(Na₂SO₄) and evaporated to dryness. The oily residue was chromatographedon a silica-gel column using hexane:ethyl acetate (8:2) as eluants.Crystallization from ethyl acetate-hexane afforded 1.2 g (25%) of pure52; mp 167-168° C. ¹H-NMR (CDCl₃): δ 1.35-1.90 (6H), 1.90-2.15 (m, 1H),2.30, 2.35 (s, 6H), 2.65 (s, 12H), 3.20 (m, 1H), 3.70 (m, 1H), 3.90 (m,1H), 5.15 (d, 1H), 6.90, 7.00 (s, 4H); ¹³C-NMR (CDCl₃): δ 20.73, 20.85,22.15, 22.37, 22.70, 26.94, 32.75, 45.34, 56.09, 70.38, 130.22, 131.57,133.98, 138.68, 139.64, 142.02, 143.10. MS-EI (m/z) 479 (M⁺), 280(M^(⊕)−199).

Compound 54: NaH (105 mg, 3.5 mmol) was added to a solution of compound52 (1.7 g, 3.5 mmol) in DMF (10 ml). The mixture was stirred at 20° C.for 30 min and a solution of compound 53 (1.34 g, 3.85 mmol) in DMF (5ml) was added in small portions. The mixture was stirred at 75° C. for 2h. The solvent was distilled off, the residue was taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(Na₂SO₄) and evaporated. The oily residue was purified by columnchromatography (hexane-ethyl acetate 8:2) which gave compound 54 (1.22g, 47%). ¹H-NMR (CDCl₃): δ 1.98 (t, 3H), 1.20-2.05 (9H), 2.20 (s, 6H),2.55, 2.65 (s, 12H), 2.70-3.55 (9H), 6.85 (s, 4H); ¹³C-NMR (CDCl₃): δ12.49, 20.80, 21.64, 21.87, 22.88, 28.72, 33.16, 36.13, 39.96, 43.80,47.95, 57.77, 61.26, 131.83, 132.94, 133.14, 138.82, 139.90, 142.07,142.63. MS-FAB (m/z) 628 (M⁺+1), 546 (M⁺−81).

Compound 55 was obtained following the procedure described for compound42. From 1.22 g (1.6 mmol) of bromoderivative 54 and 820 mg (1.76 mmol)of diamide 46, 1.26 g (77%) of tetramide 55 was obtained as a glassyoil. ¹H-NMR (CDCl₃): δ 0.80 (t, 6H), 1.20-1.75 (6H), 1.90 (m, 1H), 2.15(s, 12H), 2.35-2.60 (s, 24H), 2.65-3.40 (15H), 6.85 (b, 8H); ¹³C-NMR(CDCl₃): δ 12.38, 20.71, 22.52, 22.66, 24.72, 27.55, 28.04, 39.19,39.71, 41.02, 42.33, 42.62, 43.37, 48.81, 61.44, 131.76, 131.88, 133.10,133.89, 138.66, 139.93, 142.17, 142.33, 142.57. MS-FAB (m/z) 1012 (M⁺),828 (M⁺−184).

Compound 56 was obtained following the procedure described for compound43. From 1.26 g (1.24 mmol) of tetramide 55, 300 mg (56%) of thetetrahydrochloride 56 was obtained; mp>270° C. (decomp). ¹H-NMR (D₂O): δ1.35 (t, 6H), 1.60 (m, 1H), 1.80 (b, 3H), 2.15 (b, 6H), 2.50 (b, 1H),3.20 (m, 13H), 3.45 (m, 2H); ¹³C-NMR (D₂O): δ 13.23, 25.48, 25.73,25.79, 31.69, 31.99, 43.40, 45.91, 46.43, 46.71, 48.07, 53.20, 75.28.MS-MALDI (m/z) 285 (M⁺+1).

Compound 57: NaH (80%, 150 mg, 5 mmol) and NaBr (2.5 g, 25 mmol) wereadded to a solution of compound 52 (2.35 g, 4.9 mmol) in DMF (15 ml).The mixture was stirred at 20° C. for 30 min and a solution of1-bromoethane (2.2 g, 25 mmol) in DMF (10 ml) was added in smallportions. The mixture was stirred at 90° C. for 3 h. The solvent wasdistilled off, the residue taken up in chloroform, washed with asaturated solution of ammonium chloride, dried (Na₂SO₄) and evaporated.The product was purified by silica gel chromatography (hexane/ethylacetate 9:1). The oily residue (1.5 g, 79%) crystallized on standing; mp68-69° C. ¹H-NMR (CDCl₃): δ 1.10 (t, 3H), 1.30-2.10 (6H), 2.25 (b, 4H),2.60 (s, 6H), 3.20 (m, 2H), 3.35 (m, 2H), 3.60 (m, 2H), 6.95 (s, 2H);¹³C-NMR (CDCl₃): δ 16.35, 20.93, 21.79, 22.89, 29.32, 29.37, 36.54,38.12, 44.13, 61.40, 131.99, 132.80, 140.20, 142.52. MS-FAB 389 (M⁺+1),308 (M⁺−80).

Compound 59 was obtained following the procedure described for compound42. From 700 mg (1.80 mmol) of compound 57 and 394 mg (0.9 mmol) ofdiamide 58, 400 mg (37%) of tetramide 59 were obtained. ¹H-NMR (CDCl₃):δ 0.90 (t,6H), 1.25-1.80 (m,8H), 1.80-2.10 (m,8H), 2.15 (s, 12H), 2.40,2.50 (s, 24H), 2.60-3.35 (m, 6H), 2.85, 2.90 (s, 8H); ¹³C-NMR (CDCl₃): δ16.14, 20.85, 21.95, 21.99, 22.55, 25.49, 28.78, 28.88, 31.49, 37.87,40.50, 40.83, 43.85, 44.06, 49.30, 61.42, 131.86, 131.96, 133.09,133.40, 139.93, 139.98, 142.27, 142.40. MS-FAB (m/z) 1052 (M^(⊕)), 891(M⁺−184).

Compound 60 was obtained following the procedure described for compound43. From 400 mg (0.38 mmol) of tetramide 59, 95 mg (53%) of thetetrahydrochloride derivative were obtained; mp>270° C. (decomp.) ¹H-NMR(D₂O): δ 1.30 (t, 6H), 1.60 (m, 2H), 1.80 (m, 6H), 1.95-2.35 (6H), 2.45(m, 2H), 3.20 (m, 10H), 3.40 (m, 4H); ¹³C-NMR (D₂O): δ 13.59, 25.34,25.71, 31.75, 32.00, 43.34, 44.83, 48.02, 53.24, 64.52. MS-MALDI (m/z)325 (M⁺+1).

Compound 62: Mesitylenesulfonylchloride (3.27 g, 15 mmol) in dioxane (20ml) was added dropwise to a stirred solution of 61 (1.3 g, 10 mmol) indioxane (20 ml) and 50% KOH (15 ml) at 0° C. When addition wascompleted, the mixture was left over night at 20° C. Excess water wasadded, the solution cooled and the precipitate filtered off.Crystallization from ethylacetate-hexane gave compound 62 (2 g, 80%); mp115-116° C. ¹H-NMR (CDCl₃): δ 2.35 (s, 3H), 2.55 (t, 2H), 2.65 (s, 6H),3.25 (q, 2H), 5.15 (t, 1H), 7.0 (s, 2H); ¹³C-NMR (CDCl₃): δ 19.07,20.82, 22.78, 38.37, 117.56, 132.07, 133.0, 138.99, 142.67. MS-EI (m/z)252 (M⁺).

Compound 63: NaH (80%, 330 mg, 11 mmol) was added to a solution ofcompound 62 (2.52 g, 10 mmol) in DMF (20 ml) under N₂. The mixture wasstirred for 30 min and a solution of compound 53 (3.82 g, 11 mmol) inDMF (10 ml) was added in small portions. The mixture was stirred at 70°C. for 2 h. The solvent was distilled off, the residue taken up inchloroform, washed with a saturated solution of ammonium chloride, dried(Na₂SO₄) and evaporated to dryness. The product was purified bysilica-gel chromatography (hexane-ethyl acetate 8:2). The oily residue(3.0 g, 57%) crystallized on standing; mp 105-106° C. ¹H-NMR (CDCl₃): δ1.00 (t, 3H), 1.75 (m, 2H), 2.35 (s, 6H), 2.60 (14H), 3.10 (m, 6H), 3.45(t, 3H), 6.90, 6.95 (s, 4H); ¹³C-NMR (CDCl₃): δ 12.63, 16.94, 20.89,22.67, 25.73, 40.27, 42.19, 42.51, 44.72, 117.36, 131.95, 132.22,140.06, 140.34, 142.52, 143.33. MS-EI (m/z) 519 (M⁺), 429 (M⁺−HCN).

Compound 65: The nitrile 63 (3.0 g, 5.7 mmol) was dissolved in a mixtureof ethanol (150 ml) and concentrated hydrochloric acid (1.5 ml). PtO₂was added (300 mg), the mixture was hydrogenated at 50 psi over night,the catalyst was filtered off and the solvent evaporated to afford anoily residue of compound 64, which was used in the next step withoutfurther purification. Free base ¹H-NMR (CDCl₃): δ 1.00 (t, 3H), 1.55 (m,2H), 1.75 (m, 2H), 2.30 (s, 6H), 2.55 (14 H), 2.90-3.30 (8H), 6.95 (s,4H); ¹³C-NMR (CDCl₃): δ 12.64, 20.87, 22.69, 25.35, 30.93, 39.04, 40.12,42.65, 43.11, 131.86, 133.10, 140.04, 142.43. MS-FAB (m/z) 524 (M⁺+1).

Mesitylenesulfonylchloride (1.86 g, 8.55 mmol) in dioxane (15 ml) wasadded dropwise to a stirred mixture of 64 (3.0 g, 5.7 mmol) dissolved indioxane (30 ml) and 50% KOH (15 ml) at 0° C. The reaction mixture wasallowed to reach room temperature and was kept for further 2 h. Anexcess of water was added and the mixture was extracted with chloroform,dried (Na₂SO₄) and evaporated to dryness. Purification was achieved bysilica gel column chromatography using hexane-ethyl acetate (8:2) aseluant; 2.79 g (69%) of 65 were obtained. ¹H-NMR (CDCl₃): δ 0.95 (t,3H), 1.60 (m, 4H), 2.30 (s, 9H), 2.50 (s, 12H), 2.65 (s, 6H), 2.85 (m,2H), 3.05 (6H), 3.20 (t, 2H), 5.00 (t, 1H), 6.95 (6H); ¹³C-NMR (CDCl₃):δ 12.45, 20.81, 22.73, 25.23, 27.46, 39.19, 33.99, 42.49, 42.92, 43.17,131.84, 133.05, 133.82, 138.80, 139.90, 141.92, 142.36, 142.64. MS-FAB(m/z) 705 (M^(⊕)).

Compound 66 was obtained following the procedure described for compound42. From 705 mg (1 mmol) of 65 and 426 mg (1.1 mmol) of 57, 470 mg (46%)of tetramide 66 was obtained as a glassy product. ¹H-NMR (CDCl₃): δ0.85-1.10 (t, 6H), 1.35-2.10 (m, 11H), 2.30 (s, 12H), 2.40-2.65 (m,24H), 2.75-3.55 (m, 13H), 6.95 (m, 8H); ¹³C-NMR (CDCl₃): δ 12.64, 16.11,20.91, 22.08, 22.75, 24.81, 25.09, 28.83, 29.07, 37.93, 40.08, 40.84,42.50, 42.81, 43.11, 43.42, 49.11, 61.43. MS-FAB (m/z) 1013 (M⁺+1).

Compound 67 was obtained following the procedure described for compound43. From 470 mg (0.46 mmol) of tetramide 66, 142 mg (71%) of thetetrahydrochloride derivative was obtained; mp>250° C. (decomp). ¹H-NMR(D₂O): δ 1.30 (t, 6H), 1.60 (m, 1H), 1.85 (b, s, 3H), 2.15 (m, 6H), 2.45(m, 1H), 3.15 (m, 13H), 3.45 (m, 2H); ¹³C-NMR (D₂O): δ 13.29, 13.57,25.34, 25.44, 25.64, 31.68, 31.94, 43.27, 44.80, 45.86, 46.62, 47.42,47.97, 53.19, 64.50. MS-MALDI 285 (M⁺+1), 286 (M⁺+2).

Compound 68a: 4-Cyanobenzaldehyde (Aldrich, 1.31 g, 10 mmol) wasdissolved in 30 ml anhydrous MeOH followed by the addition of MgSO₄(anhydrous, 1.5 g) and 1,4-diaminobutane (Aldrich, 0.44 g, 5 mmol) andthe mixture was stirred under argon over night. The suspension wascooled in an ice bath and NaBH₄ (2.0 g) was added in portions andstirring continued for 2 h at 0° C. The methanol was evaporated undervacuum and the resulting solid was partitioned between 35 ml H₂O and 50ml CHCl₃. Some of the solid was not soluble in either the H₂O or theCHCl₃ and was filtered off and the aqueous layer was extracted withCHCl₃ (2×25 ml). The pooled organic layers were dried (MgSO4),evaporated and the solid was recrystallized from ethyl acetate-hexane,yield 1.1 g (35%); mp 90.6-90.8° C. ¹H-NMR (CDCl₃): δ 1.43 (broad, 2H,NH), 1.55 (m, 4H, CH₂), 2.63 (m, 4H, NCH₂), 3.85 (s, 4H, benzylic CH₂),7.44 (m, 4H, Ph), 7.60 (m, 4H, Ph); ¹³C-NMR (CDCl₃): δ 27.78, 49.28,53.44, 110.65, 118.88, 128.52, 132.12, 146.21. MS (m/z) 318 (M⁺), 185,145, 131, 116 (100%), 70.

Compound 68b was prepared from 4-cyano-benzaldehyde and1,5-diaminopentane as described above for 68a; 42% yield; mp 92.9-93.0°C. ¹H-NMR (CDCl₃): δ 1.40 (m, 4H, NH, CH₂), 1.50 (m, 4H, CH₂), 2.59 (m,4H, NCH₂), 3.83 (s, 4H, benzylic CH₂), 7.45 (m, 4H, Ph), 7.59 (m, 4H,Ph); ¹³C-NMR (CDCl₃): δ 24.86, 29.87, 49.29, 53.40, 110.50, 118.85,128.48, 132.04, 146.19. MS (m/z) 332 (M⁺), 216, 199, 145, 116 (100%),84.

Compound 68c was prepared from 4-cyanobenzyldehyde and 1,6-diaminohexaneas described above for 68a; 45% yield; mp 95.6-95.8° C. ¹H-NMR (CDCl₃):δ 1.35 (m, 4H, CH₂), 1.50 (m, 6H, NH, CH₂), 2.60 (t, J=6.92 Hz, 4H,NCH₂), 3.84 (s, 4H, benzylic CH₂), 7.44 (m, 4H, Ph), 7.60 (m, 4H, Ph);¹³C-NMR (CDCl₃): δ 27.17, 30.02, 49.42, 53.50, 110.65, 118.92, 128.55,132.14, 146.27. MS (m/z) 346 (M⁺), 230, 213, 145, 116 (100%) 98.

Compound 69a: Dinitrile 68a (0.75 g, 2.36 mmol) was dissolved inanhydrous THF, lithium bis(trimethylsilyl)amide (9.43 ml of a 1 msolution in THF) was added slowly under argon atmosphere. The mixturewas stirred at room temperature for 2 h; then cooled in an ice bath,followed by the addition of 4 equivalents of 6N HCl in ether. A whitesolid precipitated immediately and was filtered after 12 h. The solidwas recrystallized from ethanol-ether to afford 1.19 g of compound 69a(93%). ¹H-NMR (D₂O): δ 1.87 (m, 4H, CH₂), 3.22 (m, 4H, CH₂N), 4.40 (s,4H, benzylic CH₂), 7.74 (m, 4H, Ph), 7.91 (m, 4H, Ph); ¹³C-NMR(DMSO-d₆): δ 22.68, 46.09, 49.28, 128.10, 128.47, 130.69, 138.15,165.44. MS-ESI (m/z) 353.2 (M⁺), 177.2 (100%).

Compound 69b was prepared from 68b in 92% yield as described above for69a. ¹H-NMR (D₂O): δ 1.52 (m, 2H, CH₂), 1.80 (m, 4H, CH₂), 3.19 (m, 4H,NCH₂), 4.40 (s, 4H, benzylic CH₂), 7.75 (m, 4H, Ph), 7.91 (m, 4H, Ph);¹³C-NMR (DMSO-d₆): δ 24.90, 26.91, 48.96, 51.88, 130.29, 130.46, 132.43,139.51, 167.52. MS-ESI (m/z) 367.2 (M⁺), 350.2 (100%), 301.2.

Compound 69c was prepared from 68c as described above for 69a in 96%yield. ¹H-NMR (D₂O): δ 1.46 (m, 4H, CH₂), 1.78 (m, 4H, CH₂), 3.16 (m,4H, NCH₂), 4.39 (s, 4H, benzylic CH₂), 7.74 (m, 4H, Ph), 7.91 (m, 4H,Ph); ¹³C-NMR (DMSO-d₆): δ 25.24, 25.82, 46.73, 49.44, 128.35, 128.56,130.81, 138.38, 165.58. MS-ESI (m/z) 381.2 (M⁺), 191.2 (100%), 150, 116.

Compound 70: Triamide 18 (4.3 g, 5.8 mmol) was dissolved in 30 ml of DMFand 80% NaH (208 mg, 6.9 mmol) was added. The mixture was stirred undera N₂ atmosphere for 1 h and 1.12 g (7.5 mmol) of bromobutyronitriledissolved in 3 ml of DMF were added all at once. The reaction mixturewas heated for 3 h at 90° C. The solvent was distilled-off and theresidue was dissolved in chloroform and washed twice with a saturatedsolution of amonium chloride; dried (NaSO₄) and evaporated to dryness.Flash chromatography of the residue using hexane-ethyl acetate (6:4) aseluant gave the yellow oil 70 (3.7 g, 77%). ¹H-NMR (CDCl₃): δ 0.95 (t,3H), 1.35 (m, 8H), 1.85 (m, 2H), 2.20 (t, 2H), 2.30 (s, 9H), 2.55 (s,18H), 3.10 (m, 10H), 3.25 (t, 2H), 6.95 (s, 6H), MS-FAB (m/z) 823(M⁺+Na), 639, 457.

Compound 71: Nitrile 70 (3.7 g, 4.6 mmol) was dissolved in 20 ml ofchloroform and 150 ml of ethanol were added. The mixture was reducedover 0.35 g of PtO₂ at 50 psi over night. The catalyst was filtered-offand the solvent evaporated to dryness. The oily residue was dried invacuo for 2 h and dissolved in 50 ml of Cl₃CH and 12 ml 2N NaOH. Themixture was cooled in an icewater bath with efficient magnetic stirringand 1.50 g (6.9 mmol) of mesitylene chloride dissolved in 10 ml ofchloroform were added all at once. After 2 h the organic layer wasseparated, washed twice with a saturated solution of amonium chloride,dried (NaSO₄) and evaporated to dryness. Flash chromatography of theresidue using hexane-ethyl acetate (7:3) as eluant provided thetetramide 71 as a colorless oil (3.3 g, 73% over two steps). ¹H-NMR(CDCl₃): δ 0.95 (t, 3H), 1.40 (m, 12H), 2.30 (s, 12H), 2.60 (s, 24H),2.80 (b, 2H), 3.10 (m, 12H), 4.70 (b, 1H), 6.90 (s, 8H). MS-FAB (m/z)1010 (M⁺+1+Na), 826, 643.

Compound 72: The tetramide 71 (6.28 g, 6.3 mmol) was dissolved in 40 mlof DMF and 80% NaH (230 mg, 7.6 mmol) was added. The mixture was stirredunder a N₂ atmosphere for 1 h and 1.30 g (8.8 mmol) ofbromobutyronitrile dissolved in 3 ml of DMF were added all at once. Thereaction mixture was heated for 3 h at 90° C., the solvent wasdistilled-off and the residue was extracted into chloroform and washedtwice with a saturated solution of amonium chloride; dried (NaSO₄) andevaporated to dryness. Flash chromatography of the residue withhexane-ethyl acetate (7:3) as eluant provided the nitrile 72 (5.0 g,74%). ¹H-NMR (CDCl₃): δ 0.95 (t, 3H), 1.35 (m, 12H), 1.80 (m, 2H), 2.25(t, 2H), 2.35 (s, 12H), 2.70 (s, 24H), 3.10 (m, 14H), 3.25 (t, 2H), 7.0(s, 8H). MS-FAB (m/z) 1077 (M⁺+1+Na), 893, 711, 586.

Compound 73: Nitrile 72 (6.0 g, 5.6 mmol) was dissolved in 20 ml ofchloroform and 150 ml of ethanol were added. The mixture was reducedover 600 mg of PtO₂ at 50 psi overnight. The catalyst was filtered-offand the solvent evaporated to dryness. The oily residue was dried invacuo for 2 h and dissolved in 100 ml of chloroform and 15 ml 2N NaOH.The mixture was cooled in an icewater bath with efficient magneticstirring, and 1.80 g (8.4 mmol) of mesitylene chloride dissolved in 10ml of Cl₃CH was added all at once. After 2 h the organic layer wasseparated, washed twice with a saturated solution of amonium chloride,dried (Na₂SO₄) and evaporated to dryness. Flash chromatography of theresidue using hexane-ethyl acetate (7:3) as eluant gave the pentamide 73as a colorless oil (5.0 g, 71% over two steps). ¹H-NMR (CDCl₃): δ 0.95(t, 3H), 1.35 (m, 16H), 2.30 (s, 15H), 2.55 (s, 30H), 2.75 (bs, 2H),3.05 (m, 16H), 4.70 (b, 1H), 6.90 (s, 10H). MS-FAB (m/z) 1261 (M⁺−1+Na),1077, 895.

Compound 74: Pentamide 73 (3.4 g, 2.7 mmol) was dissolved in 30 ml ofDMF and 60% NaH (162 mg, 4.05 mmol) was added. The mixture was stirredunder a N₂ atmosphere for 0.5 h and 2.3 g (10.8 mmol) of 2-bromoethanolbenzylether dissolved in 3 ml of DMF were added all at once. Thereaction mixture was heated for 2 h at 80° C., the solvent wasdistilled-off and the residue was dissolved in chloroform and washedtwice with a saturated solution of amonium chloride, dried (NaSO4) andevaporated to dryness. Flash chromatography of the residue usinghexane-ethyl acetate (7:3) as eluant provided the product 74 (2.6 g,70%). ¹H-NMR (CDCl₃): δ 0.95 (t, 3H), 1.30 (m, 16H), 2.30 (s, 15H), 2.50(s, 30H), 2.90-3.20 (m, 18H), 3.25 (t, 2H), 2.35 (t, 2H), 4.35 (s, 2H),6.95 (s, 10H), 7.20-7.35 (m, 5H). ¹³C NMR (CDCl₃): δ 12.65, 20.84,22.67, 22.71, 24.41, 24.66, 39.97, 44.48, 44.88, 46.59, 68.01, 72.95,127.46, 127.57, 128.25, 131.83, 131.89, 133.28, 139.88, 139.95, 140.04,142.16, 142.23. MS-FAB (m/z) 1394 (M⁺−2+Na) 1030.

Compound 75: Pentamide 74 (1.2 g, 0.87 mmol) was dissolved in 12 ml ofmethylene chloride followed by the addition of 30% HBr/acetic acid (16ml) and phenol (3.0 g, 32 mmol). The mixture was stirred at roomtemperature overnight, water (16 ml) was added, followed by extractionwith methylene chloride (3×10 ml). The aqueous layer was evaporated invacuo. The residue was dissolved in 2N NaOH (4 ml) and 50% KOH (4 ml)followed by extraction with chloroform (4×10 ml). After removal of thesolvent the residue was dissolved in ethanol (20 ml) and acidified withconcentrated hydrochloric acid (0.5 ml). The white precipitate (75) wasrecrystallized from aqueous ethanol (440 mg, 90%); mp above 270° C.(decomp). ¹H-NMR (D₂O): δ 1.30 (t, 3H), 1.75 (b, 16H), 2.90-3.30 (m,20H), 2.85 (t, 2H). ¹³C NMR (D₂O): δ 13.29, 25.48, 25.59, 45.70, 49.04,49.49, 49.67, 51.88, 59.39. MS-MALDI (m/z) 374 (M⁺+1).

Compound 76: Pentamide 73 (850 mg, 0.68 mmol) was dissovled in DMF (15ml) and 80% NaH (30 mg, 1 mmol) was added. The mixture was stirred undera N₂ atmosphere at room temperature for 0.5 h and 137 mg (0.30 mmol) of73 dissolved in 5 ml of DMF were slowly added. The reaction mixture washeated for 2 h at 80° C., the solvent was distilled-off and the residuewas dissolved in chloroform and washed twice with a saturated solutionof amonium chloride, dried (NaSO₄) and evaporated to dryness. Flashchromatography of the residue using hexane-ethyl acetate-methanol(6:4:0.1) as eluant afforded the product 76 (590 mg, 77%). ¹H-NMR(CDCl₃): δ 0.95 (t, 6H), 1.15-1.40 (m, 32H), 2.30 (s, 30H), 2.55 (s,60H), 2.90-3.25 (m, 36H), 3.60 (d, 4H), 5.40 (t, 2H), 6.95 (s, 20H).MS-FAB 2553 (M⁺+Na).

Compound 77 was obtained following the procedure described for compound75. From 650 mg (0.25 mmol) of decamide 76, 225 mg (81%) ofdecahydrochloride 77 was obtained; mp>270° C. (decomp). ¹H-NMR (D₂O): δ1.30 (t, 6H), 1.75 (b, 32H), 3.10 (b, 36H), 3.75 (b, 4H), 6.05 (b, 2H);¹³C NMR (D₂O): δ 13.28, 25.57, 45.66, 49.00, 49.13, 49.64, 50.86,131.15. MS-ESI 711 (M⁺+1).

Compound 78 was obtained following the procedure described for compound76. From 850 mg of 73, 360 mg (47%) of decamide 78 were obtained. ¹H-NMR(CDCl₃): δ 0.95 (t, 6H), 1.15-1.45 (m, 32H), 2.30 (s, 30H), 2.55 (s,60H), 2.90-3.20 (b, 36H), 3.65 (d, 4H), 5.40 (t, 2H), 6.90 (s, 20H).MS-FAB (m/z) 2553 (M⁺+Na).

Compound 79 was obtained following the procedure described for compound75. From 330 mg (0.13 mmol) of decamide 78, 127 mg (90%) ofdecahydrochloride 79 was obtained; mp>270° C. (decomp). ¹H-NMR (D₂O): δ1.30 (t, 6H), 1.80 (b, s, 32H), 3.10 (b, 36H), 3.85 (d, 4H), 6.0 (t,2H). ¹³C NMR (D2O): δ 13.31, 25.59, 45.71, 46.83, 49.05, 49.39, 49.69,129.21. MS-ESI (m/z) 512 (M⁺+2).

Compound 96.

Pentamide 74 (1.4 g, 1.01 mmol) was dissolved in 100 ml of ethanol and200 mg of 10% Pd/C was added. The mixture was hydrogenated for 4 h at 50psi. The catalyst was filtered off and and solvent evaporated todryness. Silica-gel column chromatography using ethyl acetate/hexane 6:4as running solvent afforded 1.0 g (80%) of desired product, as an oil.¹H NMR (CDCl₃) δ: 0.95 (t, 3H), 1.30 (m, 16H), 2.30 (s, 15H); 2.55 (s,30H), 3.10 (m, 18H), 3.25 (t, 2H), 3.60 (t, 2H), 6.95, (s, 10H), ¹³C NMRδ: 12.67, 20.89, 22.75, 24.52, 40.02, 44.54, 44.97, 46.83, 48.22, 60.29,131.88, 132.78, 133.28, 139.95, 140.11, 142.33

Compound 97

Alcohol 96 (470 mg, 0.36 mmole) was dissolved in tetrahydrofuran (5 ml),Boc-Gln (97 mg, 0.39 mmole), dicyclohexylcarbodiimide (89 mg, 0.43mmole), and dimethylaminopyridine (5 mg, 0.039 mmole) were added. Thereaction mixture was stirred overnight at room temperature. Thecyclohexylurea was filtered off and the filtrate evaporated to dryness.The residue was dissolved in chloroform, washed twice with 2N HCl, oncewith water, and twice with a saturated solution of NaHCO₃, then driedand evaporated. The product was purified by silica-gel columnchromatography using methanolchloroform 2% as running solvent. The aminoacid-polyamine conjugate weighed 250 mg (45%). ¹H NMR (CDCl₃) δ: 0.95(t, 3H), 1.30 (m, 18H), 1.45 (s, 9H), 1.90-2.20 (m, 2H), 2.35 (s, 15H),2.60 (s, 30H) 2.90-3.25 (m, 18H), 3.45 (m, 2H), 4.10-4.35 (m, 3H), 6.95(s, 10H); ¹³C NMR (CDCl₃) δ: 12.57, 20.78, 22.63, 24.63, 28.19, 31.48,39.92, 44.04, 44.43, 44.82, 45.92, 53.06, 61.96, 79.80, 131.99, 133.33,139.80, 142.12, 156.40, 171.70, 174.25.

Compound 98

Amino acid-polyamine conjugate 97 (170 mg, 0.11 mmole) was treated withtrifluoroacetic acid (1.25 ml) in methylene chloride (5 ml) for 30minutes. The solvent was evaporated at room temperature, the residue wasdissolved in chloroform and washed with a saturated solution of NaHCO₃,then dried and evaporated to dryness. After drying in vacuo, the residueweighted 158 mg (100%) and was used in the next step without furtherpurification.

The deprotected amino acid-polyamine conjugate (158 mg, 0.11 mmole) wasdissolved in tetrahydrofuran (5 ml), Boc-Leu (30 mg, 0.13 mmole),dicyclohexylcarbodiimide (27 mg, 0.14 mmole) and dimethylaminopyridine(16 mg, 0.13 mmole) were added. The reaction mixture was stirredovernight at room temperature. The cyclohexylurea was filtered off andthe filtrate evaporated to dryness. The residue was dissolved inchloroform, washed twice with 2N HCl, once with water, and twice with asaturated solution of NaHCO₃, dried and evaporated. Thedipeptide-polyamine conjugate was purified by silica-gel columnchromatography using methanol/chlorofom 4% as running solvent to yield130 mg (70%). ¹H NMR (CDCl₃) δ: 0.95 (m, 9H), 1.30 (m, 16H), 1.45 (s,9H), 1.50-2.05 (m, 3H), 2.30 (s, 19H), 2.60 (s, 30H), 3.50 (m, 2H)3.90-4.30 (m, 3H), 4.50 (m, 1H), 6.95 (s, 10H).

Compound 99 was obtained following the procedure described for compound21. From 100 mg (0.061 mmol) of dipeptide-polyamine conjugate 98, 26 mg(50%) of the hexachloride 99 was obtained; mp>270° C. (decomp). ¹H NMR(CDCl₃) δ: 0.95 (m, 6H), 1.30 (t, 3H), 1.40-1.90 (m, 20H), 1.90-2.50 (m,3H), 3.0-3.30 (m, 20H), 3.40-4.20 (m, 4H). ESI-MS (m/z) 615 (M⁺+1), 651(M⁺+1+HCl), 687 (M⁺+1+2HCl)

Example 2 Synthesis of Novel Quinone Derivatives Synthetic Preparationof Quinone Compounds

Preparation of quinones of the invention is described below and depictedin the Figures. New chemistry was developed in order to construct drugswhere the 1,2-naphthoquinone moiety is bound to a DNA minor groovebinder unit or a DNA intercalator. While not wishing to limit theinvention to any particular theory of operation, it is believed that the1,2-naphthoquinone derivatives “poison” topoisomerase II and transformthis essential DNA replication enzyme into a nuclease-type enzyme thatcleaves DNA. It is postulated that this modification of topoisomerase IIby the 1,2-naphthoquinones is very likely due to the alkylation of thethiol residues of the enzyme by the quinones (Michael additions). Scheme501 outlines derivatization reactions leading to 1,2-naphthoquinoneintermediates. The silver salt of 2-hydroxy-1,4-naphthoquinone wasalkylated with the tert-butyl or benzyl esters of 5-bromo-pentanoic acidto give either 501 or 502. The benzyl ester 502 was transformed into theacid 503 by hydrogenolysis. The silver salt was also alkylated with6-bromohexanol to give 504, or with 1,6-diiodohexane to give 505. Thealcohol 504 treated with triphosgene gives 506 (Scheme 502). The acid503 can be derivatized by reaction with3-amino-1-methyl-5-methyloxycarbonylpyrrole (Baird and Dervan (1996) J.Am. Chem. Soc. 118:6141) in the presence ofo-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU) and diisopropylethyl amine (DIEA) to give the amide 507. Thesilver salt of 2-hydroxy-1,4-naphthoquinone reacted with pivalylchloride to give 508 (Scheme 502). Acid 503 was condensed with thepolypyrrole amide 509 (Baird and Dervan (1996) J. Am. Chem. Soc.118:6141) after cleavage of the protecting t-butyl group with TFA. Theresulting product 510 is a molecule where the 1,2-naphthoquinone moietyis covalently bound to a DNA minor groove binder (Scheme 503). Alcohol504 was condensed using the Mitsonobu reaction (triphenylphosphine,diethyl acetylenedicarboxylate) with 4-hydroxy-benzonitrile to give 511.Iodide 505 was reacted with the tetrabutyl ammonium salt of phenol togive 512. The acid 503 was esterified with 3-dimethylaminophenol usingdicyclohexylcarbodiimide (DCC) and 4-dimethylamino pyridine (DMAP) andgave 513. By reaction of 505 and the tetrabutylammonium salt of Hoechst33528 it was possible to obtain 514, where the quinone is covalentlybound to the DNA minor groove binder. By esterification of 504 with6-aminohexanoic acid (used as its BOC derivative and deprotected withTFA) in the presence of DCC and DMAP, it was possible to obtain 515 asits trifluoroacetate (Scheme 504). By condensation of the acid 503 withthe N-ethyl diamide 516, the polyamide quinone 517 was prepared (Scheme504).

A new class of 4-aminoalkyl substituted 1,2-naphthoquinones was obtainedfollowing the outline depicted in Scheme 505. A Vilsmeier reaction on1,2-dimethoxynaphthalene gave the formyl derivative 518. It wasconverted by reductive amination with n-butylamine into 519. Treatmentof 519 with acetyl chloride gave 520, while treatment withtrifluoroacetic anhydride gave 521 (Scheme 505). Acylation of 519 withmorpholino succinyl chloride gave 522. Cleavage of the 1,2-dimethoxygroups of 519 with boron tribromide gave the quinone 523 which was foundto exist in the p-quinonemethine form. Cleavage of the dimethoxyresidues of 520 and 521 led to the expected quinones 524 and 525.Cleavage of the methoxy residues of 522 gave the quinone 526 (Scheme505).

The 1,2-naphthoquinone residue was also covalently bound to a porphyrinbackbone, since porphyrins are known to concentrate in cancer tissues.By reaction of the iodide 505 with the tetrabutylammonium salt ofmeso-p-hydroxyphenylporphyrin, the porphyrin quinone 527 was obtained(Scheme 506).

By esterification of4,4′,4″,4″′-(21H,23H-porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)with the quinone alcohol 504 in the presence of EDCI (1,(3-dimethylaminopropyl)-3-ethylcarbodiimide) and DMAP it was possible to preparethe quinone-porphyrin 528 (Scheme 507).

Synthesis of 1,2-naphthoguinones Bound to DNA Intercalators

It is known that 4-aminoacridine derivatives intercalate in the DNAhelix. Therefore syntheses of 1,2-naphthoquinone residues bound to4-aminoacridine derivatives were designed (Scheme 508). The salt(6-hydroxyhexyl)triphenylphosphonium bromide was prepared by thereaction of 6-bromohexanol with triphenylphosphine in refluxingacetonitrile. Wittig reaction of (6-hydroxyhexyl)triphenylphosphoniumbromide with 4-acetamidobenzaldehyde produced alkene 529 as a mixture ofE and Z isomers. Reduction of the double bond (H₂, Pd/C) and acidichydrolysis (2N HCl, MeOH) afforded 4-(7-hydroxyheptyl)-aniline 530.Aniline 530 was reacted with 9-chloroacridine in MeOH in the presence oftriethylamine to give alcohol 531. Alcohol 531 was converted to iodide532 by reaction with methanesulfonyl chloride in pyridine, followed byreaction with sodium iodide in acetone. Reaction of iodide 532 with thesilver salt of 2-hydroxy-1,4-naphthoquinone afforded quinone 533 as amixture of ortho- and para-quinone isomers. The ortho- and para-quinoneisomers could be separated and purified by column chromatography.

A second approach to these types of compounds is shown in Scheme 509.The isomer mixture 534 was converted to the iodide 535 by reaction withmethanesulfonyl chloride in CH₂Cl₂ in the presence of pyridine, followedby a displacement with sodium iodide in acetone. Reaction of 535 withtriphenylphosphine in refluxing acetonitrile afforded the phosphoniumsalt. A Wittig reaction between the phosphonium salt and naphthaldehyde518 produced diene 536 (as a mixture of double bond isomers). Reductionwith H₂ over Pd/C followed by hydrolysis (2N HCl, MeOH) gave aniline537. Aniline 537 was reacted with 9-chloroacridine in MeOH in thepresence of triethylamine to give 538. Cleavage of the methyl etherswith boron tribromide gave quinone 539.

A third synthetic approach to a 1,2-naphthoquinone moiety bound to anaminoacridine intercalator is depicted in Scheme 510. Aminoacridine wasprotected with mesitylenesulfonyl chloride to give 541, which was thenalkylated with 1,5-dibromopentane to 542. The latter is brought intoreaction with the silver salt of 2-hydroxy-1,4-naphthoquinone and thequinone-acridine 543 was thus obtained. Cleavage of the amide groupusing samarium iodide gave 544, the expected compound.

Synthesis of 1,2-naphthoquinol Phosphates

In order to obtain 1,2-naphthoquinone derivatives that behave as“pro-drugs” the synthesis of quinol phosphates that can be hydrolyzed bycell phosphatases to liberate the parent quinones was carried out.Scheme 511 outlines the synthesis of the quinol phosphates. The parent1,2-naphthoquinone 546 was brought into reaction with dibenzylphosphiteto give a mixture of the two possible regioisomers 547. By cleavage ofthe benzyl residues with hydrogen in the presence of 10% Pd on charcoalthe mixture of the two possible quinol phosphates 548 was obtained. Theywere used as such in the biological studies.

Synthesis of 8-hydroxy-β-lapachone 555

Scheme 512 outlines the synthesis of 555, a phenol derivative ofβ-lapachone that could be used as a building block for the constructionof peptide derivatives of β-lapachone. The synthesis starts with thecommercially available ester 549, that is acetylated using aFriedel-Crafts reaction to give 550. Cyclization of 550 in the presenceof base and air gave the p-quinone 551. Alkylation of 551 with dimethylallyl bromide gave a mixture of the C-alkyl derivative 552 and theO-alkyl derivative 553. They were separated and on treatment of 552 withconcentrated sulfuric acid, the 8-methoxy-β-lapachone 554 was obtained.Cleavage of the methoxy group with boron tribromide gave the expectedσ-naphthoquinone 555.

Synthesis of 1,2-naphthoguinone Bisulfite Adducts

Bisulfite adducts of 1,2-naphthoquinones were prepared as “pro-drugs.”They are stable in aqueous solutions at pH below 7 but liberate thequinone core at pH above 7. Since biological media are usually above pH7, the bisulfite adducts led to a slow release of the quinones afteradministration in an aqueous medium. General preparation procedures aregiven in the Experimental section.

Synthesis of 1,2-naphthocluinone Peptides

1,2-Naphthoquinone conjugates of tetra and hexapeptides were prepared toobtain “prodrug” derivatives that can be cleaved by prostatic PSA. Theguidelines followed for the synthesis of the peptides were based on thepublished results of Isaacs and coworkers (Denmeade et al. Cancer Res.1997, 57, 4924), where they define the substrate specificity of PSA(prostate specific antigen). The synthesis of a quinone tetrapeptide isoutlined in Scheme 513 for the 3-β-alanyloxy-β-lapachone (SL-11006)conjugate. SL-11006 (Quin) was coupled to Boc-Gln with DCC in thepresence of 1-hydroxybenzotriazole to give Boc-Gln-Quin. Removal of theBoc group from Boc-Gln-Quin with TFA in CH₂Cl₂ gave TFA.Gln-Quin.Boc-Leu was coupled to TFA.Gln-Quin with DCC in the presence of1-hydroxybenzotriazole to give Boc-Leu-Gln-Quin. Removal of the Bocgroup from Boc-Leu-Gln-Quin with TFA in CH₂Cl₂ gave TFA.Leu-Gln-Quin.Boc-Lys(Nε-Cbz) was coupled to TFA.Leu-Gln-Quin with DCC in the presenceof 1-hydroxybenzotriazole to give Boc-Lys(Nε-Cl-Cbz)-Leu-Gln-Quin.Removal of the Boc group from Boc-Lys(Nε-Cbz)-Leu-Gln-Quin with TFA inCHCl₃ gave TFA. Lys(Nε-Cbz)-Leu-Gln-Quin. Morpholino-Ser(OBn) wascoupled to TFA-Lys(Nε-Cbz)-Leu-Gln-Quin with DCC in the presence of1-hydroxybenzotriazole to givemorpholino-Ser(OBn)-Lys(Nε-Cbz)-Leu-Gln-Quin. The side chain protectinggroups were removed by hydrogenolysis to yieldmorpholino-Ser-Lys-Leu-Gln-Quin. During the hydrogenolysis, the quinonewas reduced to the hydroquinone, which reoxidized to the quinone onexposure to air. Morpholino-Ser(OBn) was prepared from N-Fmoc-Ser(OBn).Esterification of N-Fmoc-Ser(OBn) with isobutylene in the presence of acatalytic amount of H₂SO₄ afforded N-Fmoc-Ser(OBn)-OtBu. The Fmoc groupwas removed with piperidine in CH₂Cl₂ to produce Ser(OBn)-OtBu. Reactionof Ser(OBn)-OtBu with 4-morpholinecarbonyl chloride in pyridine yieldedmorpholino-Ser(OBn)-OtBu. Morpholino-Ser(OBn)-OtBu was hydrolyzed withTFA in CH₂Cl₂ to yield morpholine-Ser(OBn).

The synthesis of a tetrapeptide conjugate of 3-leucyloxy-β-lapachone isoutlined in Scheme 514.

EXPERIMENTAL

tert-Butyl δ-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]valerate (501). Amixture of tert-butyl 5-bromovalerate (1 g, 4.2 mmol) and the silversalt of 2-hydroxy-1,4-naphthoquinone (0.8 g, 3.84 mmol) in benzene (10mL), was stirred for 24 h at 50° C. The reaction mixture was filteredthrough celite and the solvent was removed in vacuo. The residue waspurified by flash chromatography (5% methanol in chloroform) to give ayellow solid (384 mg, 30%). ¹H NMR (CDCl₃) 8.12 (d, J=7.7 Hz, 1H), 7.89(d, J=7.7 Hz, 1H), 7.70 (t, J=6.1 Hz, 1H), 7.59 (t, J=6.4 Hz, 1H), 5.95(s, 1H), 4.17 (t, J=5.9 Hz, 2H), 2.35 (t, J=7.2 Hz, 2H), 1.90-2.05 (m,2H), 1.78-1.90 (m, 2H), 1.47 (s, 9H).

Benzyl 5-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]valerate (502). Amixture of benzyl 5-bromovalerate (2.27 g, 8.4 mmol) and the silver saltof 2-hydroxy-1,4-naphthoquinone (1.63 g, 5.81 mmol) in benzene (8 mL)was stirred for 48 h at 55° C. and filtered through celite. The filtratewas diluted with diethyl ether, extracted with a 20% aqueous solution ofNaHSO₃ then basified to pH 10-11 with Na₂CO₃, and extracted with CH₂Cl₂.Yellow solid (1.334 g, 63%). ¹H NMR (CDCl₃) 8.12 (d, J=7.5 Hz, 1H), 7.85(d, J=7.7 Hz, 1H), 7.68 (t, J=7.5, 1H), 7.58 (t, J=7.7 Hz, 1H),7.25-7.50 (m, 5H), 5.93 (s, 1H), 5.14 (s, 2H), 4.15 (t, J=5.7 Hz, 2H),2.50 (t, J=7.0 Hz, 2H), 1.8-2.2 (m, 4H).

5-[(1,2-Dioxo-1,2-dihydronaphth-4-yl)oxy]valeric Acid (503). Benzylester 502 (1.90 g, 5.22 mmol) was hydrogenated at 30 psi with Pd (400mg) in ethyl acetate (120 mL) for 6 h. The catalyst was removed byfiltration through celite, the solvent was evaporated in vacuo and theresidue was oxidized with Ag₂O (1.45 g, 6.25 mmol) in Et₂O by stirringfor 10 h. Following filtration and evaporation of the solvent theproduct was crystallized from benzene to afford 0.53 g of pure material.The mother liquor was purified by flash chromatography (CH₂Cl₂/MeOH15:1), the product dissolved in CH₂Cl₂, extracted with aqueous NaHCO₃solution, acidified to pH 1 with 3% HCl and extracted back with CH₂Cl₂to give additional 0.25 g of pure material (total yield 55%), mp134-136° C.; ¹H NMR (CDCl₃) 8.12 (d, J=7.0 Hz, 1H), 7.87 (d, J=7.6 Hz,1H), 7.70 (t, J=7.5 H), 7.59 (t, J=7.4 Hz, 1H), 7.27 (s, 1H), 4.18 (t,J=5.9 Hz, 2H), 2.51 (t, J=7.0 Hz, 2H), 1.75-2.15 (m, 4H).

1,2-Dihydro-4-(6-hydroxyhexyloxy)-1,2-dioxo-naphthalene (504). A mixtureof 6-bromohexanol-1 (4.5 g, 24.85 mmol) and the silver salt of2-hydroxy-1,4-naphthoquinone (6.46 g, 23.01 mmol) in benzene (24 mL) wasstirred for 48 h at 60° C. The reaction mixture was worked up asdescribed for 502 and crystallized from hexane to afford a yellow solid(3.18 g, 50%). mp 96-98° C., ¹H NMR (CDCl₃) 8.12 (d, J=7.5 Hz, 1H), 7.87(d, J=7.7 Hz, 1H), 7.70 (t, J=7.5 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 5.95(s, 1H), 4.15 (t, J=6.3 Hz, 2H), 3.69 (t, J=6.2 Hz, 2H), 1.92-1.97 (m,2H), 1.3-1.8 (m, 7H)

1,2-Dihydro-4-(6-iodohexyloxy)-1,2-dioxonaphthalene (505). A mixture of1,6-diiodohexane (10.14 g, 30 mmol) and the silver salt of2-hydroxy-1,4-naphthoquinone (2.81 g, 10 mmol) in benzene (60 mL) wasstirred for 12 h at room temperature. The reacton mixture was filteredthrough Celite, concentrated in vacuo, and purified by flashchromatography (hexane/EtOAc 4:1) to give a yellow solid (2.19 g, 57%);mp 85-87° C.; ¹H NMR (CDCl₃) 8.12 (dd, J=6.5, 1.0 Hz, 1H), 7.86 (dd,J=6.9, 0.9 Hz, 1H), 7.70 (dt, J=7.6, 1.5 Hz, 1H), 7.58 (dt, J=7.5, 1.3Hz, 1H), 5.95 (s, 1H), 4.15 (t, J=6.3, 2H), 3.22 (t, J=6.9 Hz, 2H),1.80-2.05 (m, 4H), 1.45-2.10 (m, 4H).

bis [6-[(1,2-Dihydro-1,2-dioxonaphth-4-yl)oxy]hexyl]carbonate (506).Pyridine (0.12 ml, 1.5 mmol) was added to a stirred solution of thealcohol 504 (200 mg, 0.73 mmol) and bis(trichloromethyl)carbonate (40mg, 0.134 mmol) in CH₂Cl₂ (5 mL) at 0° C. The cooling bath was removed,the reaction mixture was diluted with CH₂Cl₂, washed with 3% HCl, brine,dried (Na₂SO₄) and purified by column chromatography (benzene/EtOAc 4:1,2:1). The product was triturated with Et₂O to afford a yellow solid (127mg, 30%), mp 78-82° C. (decomp.). MS (LSIMS, 3-NBA) 576 (M⁺+2), 401,175; ¹H NMR (CDCl₃) 8.09 (dd, J=6.0, 1.6 Hz, 1H), 7.85 (dd, J=7.8, 1.2Hz, 1H), 7.71 (t, J=6.9 Hz, 1H), 7.58 (t, J=6.2 Hz, 1H), 5.94 (s, 1H),4.17 (t, J=6.0 Hz, 2H), 4.15 (t, J=5.6 Hz, 2H), 1.85-2.10 (m, 2H),1.65-1.85 (m, 2H), 1.40-1.65 (m, 4H).

N-(1-Methyl-5-methyloxycarbonylpyrrol-3-yl)-5-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]valeramide(507). A solution of an acid 503 (334 mg, 1.22 mmol) in DMF (1.67 mL)was treated with HBTU (462 mg, 1.22 mmol) followed by DIEA (452 mg, 3.5mmol) and stirred for 5 min. 3-Amino-1-methyl-5-methyloxycarbonylpyrrolhydrochloride (232 mg, 1.22 mmol) and DIEA (378 mg, 3 mmol) were addedto the reaction mixture. The latter was stirred for 2 h, diluted withEt₂O, the precipitate was removed, dissolved in CHCl₃, washed with 3%HCl, H₂O, aqueous NaHCO₃, H₂O again, dried (MgSO₄) and purified bychromatography on alumina column (CHCl₃/MeOH 80:1, 50:1). The productwas triturated with Et₂O/CHCl₃ to obtain a yellow-red solid (200 mg,40%); mp 122-123° C. (decomp.): MS (LSIMS, 3-NBA) 410 (M⁺), 237(M⁺−173). ¹H NMR (CDCl₃) 8.08 (d, J=7.5 Hz, 1H), 7.86 (d, J=7.3 Hz, 1H),7.68 (t, J=7.5 Hz, 1H), 7.57 (t, J=7.5 Hz, 1H), 7.38 (d, J=1.8 Hz, 1H),7.34 (s, 1H), 6.65 (d, J=2 Hz, 1H), 5.95 (s, 1H), 4.19 (t, J=5.53 Hz,2H), 3.88 (s, 3H), 3.80 (s, 3H), 2.46 (t, J=6.6 Hz, 2H), 1.90-2.15 (m,4H).

4-(tert-Butylcarbonyloxy)-1,2-dihydro-1,2-dioxonaphthalene (508). Amixture of the silver salt of 2-hydroxy-1,4-naphthoquinone (842 mg, 3mmol), and pivaloyl chloride (434 mg, 3.6 mmol) in benzene (5 mL) wasstirred for 8 h at room temperature. The reaction mixture was filteredthrough Celite, the precipitate washed with EtOAc, and the combinedorganic solutions were concentrated in vacuo and purified by flashchromatography (EtOAc/hexane 1:10, 1:5). The product was recrystallizedfrom hexane to afford a yellow solid (190 mg, 25%); mp 125-126° C.; ¹HNMR (CDCl₃) 8.15(dd, J=7.7, 1.1 Hz, 1H), 7.71(dt, J=7.7, 1.5 Hz, 1H),7.59 (dt, J=7.5, 1.2 Hz, 1H), 7.57 (dd, J=7.6, 1.1 Hz, 1H), 6.48 (s,1H), 1.44 (s, 9H).

N-[3-(Dimethylamino)propyl][3-[[3-[[3-[4-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]butylcarbonylamino]-1-methylpyrrol-5-yl]carbonylamino]-1-methylpyrrol-5-yl]carbonylamino]-1-methylpyrrol-5-yl]carboxamide(510) was prepared from acid 503 (61 mg, 0.222 mmol) and Boc-protectedpyrrolylamine 509 (84 mg, 0.148 mmol) using the procedure described for507. After the reaction was completed, the reaction mixture was dilutedwith Et₂O, the precipitate was removed, triturated with hot EtOAc andcrystallized from a CHCl₃/Et₂O mixture. The product was a yellow solid(30 mg, 28%); mp 159-162° C. (decomp.); ¹H NMR (DMSO-d₆) 9.90 (s, 1H),9.89 (s, 1H), 9.86 (s, 1H), 8.08 (bs, 1H), 7.97 (d, J=7.7 Hz, 1H), 7.87(d, J=7.2 Hz, 1H), 7.81 (t, J=7.9 Hz, 1H), 7.68 (t, J=7.2, 1H), 7.24 (s,1H), 7.19 (s, 1H), 7.04 (s, 1H), 6.89 (s, 1H), 6.84 (s, 1H), 6.06 (s,1H), 4.25 (t, J=5.8 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H),3.12-3.30 (m, 2H), 2.25-2.45 (m, 4H), 2.19 (s, 6H), 1.72-2.00 (m, 4H),1.60-1.70 (m, 2H).). MS (LSIMS, 3-NBA) 725.2 (M⁺+1).

1,2-Dihydro-4-[6-[(4-cyanophenyl)oxy]hexyloxy]-1,2-dioxonaphthalene(511). A mixture of 4-hydroxybenzonitrile (87 mg, 0.73 mmol),naphthoquinone 504 (200 mg, 0.73 mmol), PPh₃ (191 mg, 0.73 mmol) indioxane (10 mL) was cooled to 10° C. and treated with DEAD (140 mg, 0.80mmol). The reaction mixture was stirred for 10 h, concentrated in vacuoand purified by chromatography (5% EtOAc in benzene) to afford 511 as ayellow solid (171 mg, 53%), ¹H NMR (CDCl₃) 8.13 (dd, J=7.3, 1.4 Hz, 1H),8.86 (dd, J=7.7, 1.1 Hz, 1H), 7.67 (dt, J=7.5, 1.5 Hz 1H), 7.60 (dt,J=7.5, 1.5 Hz, 1H), 7.57 (d, J=8.8 Hz, 2H), 6.93 (d, J=8.9 Hz, 2H),5.96(s, 1H), 4.17 (t, J=6.4 Hz, 2H), 4.03 (t, J=6.3 Hz, 2H), 1.80-2.05 (m,4H), 1.58-1.68 (m, 4H).

1,2-Dihydro-4-[6-(phenyloxy)hexyloxy]-1,2-dioxonaphthalene (512). Phenol(28 mg, 0.3 mmol) was treated with tetrabutylammonium hydroxide (0.3 mLof 1.0 M solution in methanol) and the reaction mixture was concentratedto dryness in vacuo. Iodonaphtoquinone 505 (115 mg, 0.3 mmol) in DMF (3mL) was added to the tetrabutylammonium salt, stirred for 48 h andquenched with H₂O (10 mL). The product was extracted with CHCl₃, theextract was washed with H₂O, then brine, dried (Na₂SO₄), and purified bychromatography (5% EtOAc in benzene) to give 512 as a yellow solid (45mg, 43%) ¹H NMR (CDCl₃) 8.13 (d, J=7.4 Hz, 1H), 7.86 (d, J=7.4 Hz, 1H),7.67 (t, J=7.6 Hz, 1H), 7.61 (t, J=7.5 Hz, 1H), 7.15-7.40 (m, 2H),6.85-7.10 (m, 3H), 5.96 (s, 1H), 4.17 (t, J=6.5 Hz, 2H), 3.99 (t, J=6.2Hz), 1.70-2.10 (m, 4H), 1.35-1.70 (m, 4H).

3-Dimethylaminophenyl 5-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]valerate(513). A mixture of acid 503 (137 mg, 0.5 mmol), 3-dimethylaminophenol(82 mg, 0.6 mmol), DCC (103 mg, 0.5 mmol), and DMAP (12 mg, 0.01 mmol)in THF (2 mL) was stirred for 2 h. The reaction mixture was concentratedin vacuo, the residue dissolved in benzene, washed with H₂O and dried(Na₂SO₄). Column chromatography (10% EtOAc) in benzene gave 513 as ayellow solid (70 mg, 36%), ¹H NMR (CDCl₃) 8.13, (d, J=7.3 Hz, 1H), 7.90(d, J=7.4 Hz, 1H), 7.69 (t, J=6.1 Hz, 1H), 7.58 (t, J=7.6 Hz, 1H), 7.22(dd, J=8.1, 8.1 Hz, 1H), 6.30-6.70 (m, 2H), 5.96 (s, 1H), 4.21 (t, J=5.6Hz, 2H), 2.69 (t, J=6.5 H, 2H), 1.90-2.15 (m, 4H).

2′-[4-[6-(1,2-Dihydro-1,2-dioxo-naphth-4-yl)oxyhexyl]oxyphenyl]-5-(4-methylpiperazin-1-yl)-2,5′-bi-1H-benzimidazole(514). Hoechst 33258 (3.0 g, 5 mmol) was dissolved in a hot mixture ofisopropanol-water (24 mL/12 mL) and neutralized with ammonium hydroxide(3 mL). The precipitate was filtered, triturated with Et₂O and dried invacuo to obtain the free base of bisbenzimidazole. A 1.0 M solution ofBu₄NOH in MeOH (0.6 mL, 0.6 mmol) was added to the solution ofbisbenzimidazole (1.635 g, 3.85 mmol) in MeOH (30 mL), stirred for 15min and concentrated to dryness in vacuo. Iodonaphthoquinone 505 (1.485g, 3.87 mmol) in DMF (30 mL) was added to the tetrabutyl ammonium saltand the mixture was stirred for 48 h. The reaction mixture was suspendedin H₂O, the crude product was filtered, washed with H₂O, dried andpurified by flash chromatography (MeOH/CHCl₃ 1:9, 1:5) to afford 514 asa yellow solid (790 mg, 30%). ¹H NMR (CDCl₃/MeOH-d₄) 8.21 (s, 1H), 8.09(d, J=7.6 Hz, 1H), 8.05 (d, J=8.7 Hz, 2H), 7.85-7.95 (m, 2H), 7.48-7.75(m, 4H), 7.14 (bs, 1H), 7.10-6.98 (m 3H), 4.21 (t, J=6.3 Hz, 2H), 4.08(t, J=6.2 Hz, 2H), 2.65-2.75 (m, 4H), 2.40 (s, 3H), 1.80-2.15 (m, 4H),1.60-1.75 (m, 4H). MS (LSIMS, 3-NBA) 725.2 (M⁺+1).

Trifluoroacetate of 6-[1(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]hexyl6-aminohexanoate (515). [6-(tert-Butyloxycarbonyl)amino]hexanoic acid(139 mg, 0.6 mmol) was added into solution of DCC (113 mg, 0.55 mmol)and DMAP (64 mg, 0.52 mmol) in CH₂Cl₂ (10 mL) at 0° C. and stirred for15 min, when naphthoquinone 504 (137 mg, 0.5 mmol) was added. Thereaction mixture was stirred for 12 h at room temperature, diluted withCH₂Cl₂, extracted 3 times with an aqueous solution of KHSO₄, then with aNaHCO₃ solution followed by brine, dried (MgSO₄), and finally it wasconcentrated to dryness in vacuo and triturated with Et₂O. The residuewas dissolved in CH₂Cl₂ (3 mL), TFA (0.5 mL) was added to the solutionand the mixture stirred at 0° C. for 1 h. All volatiles were removed invacuo and the residue was triturated in Et₂O to give 515 (100 mg, 40%).as a dark yellow oil. ¹H NMR (CDCl₃) 8.90 (d, J=7.6 Hz, 1H), 7.99 (bs,3H), 7.87 (d, J=7.8 Hz, 1H), 7.71 (t, J=7.6 Hz, 1H), 7.59 (t, J=7.5 Hz,1H), 5.96 (s, 1H), 4.17 (t, J=6.3 Hz, 2H), 4.09 (t, J=6.2 Hz, 2H),2.90-3.15 (m, 2H), 2.29 (t, J=7.1 Hz, 2H), 1.90-2.10 (m, 2H), 1.30-1.85(m, 12H).

1,2-Dihydro-1,2-dioxo-4-[4-[2-[3-[2-(Ethylaminocarbonyl)ethylaminocarbonyl]propyl=aminocarbonyl]ethylaminocarbonyl]butyloxy]naphthalene(517). Acid 503 (137 mg, 0.5 mmol) was dissolved in DMF (1 mL), treatedwith HBTU (190 mg, 0.5 mmol) followed by DIEA (260 μL, 1.5 mmol) andstirred for 10 min.N-Ethyl[2-[3-(2-aminoethylcarbonylamino)propylcarbonylamino]ethyl]carboxamidehydrochloride 516 (154 mg, 0.5 mmol) and DIEA (260 μL, 1.5 mmol) wereadded to the reaction mixture, the latter was stirred for 2 h, and thereaction mixture was diluted with Et₂O. The product was filtered andtriturated with CHCl₃ to afford a yellow solid (100 mg, 38%), mp145-170° C. (decomp.) ¹H NMR (CDCl₃, MeOH-d₄) 8.10 (dd, J=7.6, 1.4 Hz,1H), 7.92 (dd, J=7.8, 1.2 Hz, 1H), 7.72 (dt, J=7.7, 1.2 Hz, 1H), 7.62(dt, 7.6, 1.3 Hz, 1H), 7.30-7.50 (m, 2H), 7.15 (bs, 1H), 5.97 (s, 1H),4.20 (t, J=5.8 Hz, 2H), 3.35-3.50 (m, 4H), 3.10-3.30 (m, 4H), 3.32-3.42(m, 4H), 2.30 (t, J=6.9 Hz, 2H), 2.19 (t, J=7.4 Hz, 2H), 1.75-2.05 (m,4H), 1.78 (t, J=7.2, 2H), 1.13 (t, J=7.3, 3H). MS (FAB, NaI) 551.2(M+Na), 529 (M⁺+1).

3,4-Dimethoxy-1-naphthaldehyde (518). A mixture of1,2-dimethoxynaphthalene (0.74 g, 4 mmol) and DMF (0.8 mL, 10 mmol) indichlorobenzene (0.8 mL) was stirred with POCl₃ at 100° C. for 2 h. Thereaction mixture was cooled to 0° C., quenched with a cold aqueoussolution of NaOAc, diluted with H₂O and extracted with benzene. Theextracts were dried (MgSO₄), concentrated and in vacuo anddichlorobenzene was removed by kugelrohr distillation at 110° C./0.5 mmHg. Column chromatography (20% EtOAc in hexane) gave the product 518(596 mg, 68%), which was used in the following step without furtherpurification. ¹H NMR (CDCl₃) 10.42 (s, 1H), 9.00-9.15 (m, 1H), 8.15-8.30(m, 1H), 7.61 (s, 1H), 7.50-7.65 (m, 2H), 4.12 (s, 3H), 4.07 (s, 3H).

4-Butylaminomethyl-1,2-dimethoxy-naphthalene (519). A suspension of PtO₂(40 mg) in EtOH (2 mL) was stirred with H₂ at 25 psi for 30 min.Naphthaldehyde 518 (596 mg, 2.8 mmol) was dissolved in EtOH and addedinto the suspension followed by the addition of butylamine (219 mg, 3mmol). The reaction mixture was hydrogenated for 6 h at 50 psi. Thecatalyst was filtered through Celite, washed with acetone and thefiltrate was concentrated to dryness to give 519 as an oil (665 mg,87%). The product was utilized in the following step without furtherpurification. ¹H NMR (CDCl₃) 8.16 (d, J=7.5 Hz, 1H), 7.99 (d, J=7.6 Hz,2H), 7.40-7.60 (m, 2H), 7.35 (s, 1H), 4.19 (s, 2H), 4.00 (s, 3H), 3.98(s, 3H), 2.76 (t, J=7.0 Hz, 2H), 1.64 (bs, 1H), 1.45-1.60 (m, 2H),1.30-1.45 (m, 2H), 0.93 (t, J=7.2 Hz, 3H).

4-(N-Acetyl-N-butylaminomethyl)-1,2-dimethoxy-naphthalene (520).Triethylamine (350 μL, 2.5 mmol) was added to a solution ofaminonaphthalene 519 (250 mg, 0.9 mmol) and AcCl (90 μL, 1.27 mmol) inCH₂Cl₂ (5 mL) at 0° C. The cooling bath was removed after 10 min, thereaction mixture was stirred for 1 h at room temperature, dilutedfivefold with CH₂Cl₂, washed with an aqueous solution of NaHCO₃ followedby 3% HCl, brine and dried (MgSO₄). The crude product (315 mg, 100%)obtained after evaporation of the solvent was used in the following stepwithout further purification. ¹H NMR (CDCl₃) 8.19, 8.15 (2d, J=7.6, 8.4Hz, 1H), 8.97, 7.80 (2d, J=7.9, 8.2 Hz, 1H), 7.35-7.58 (m, 2H), 7.16,7.04 (2s, 1H), 5.05, 4.95 (2s, 2H), 4.01, 3.99 (2s, 3H), 3.99, 3.96 (2s,3H), 3.47, 3.13 (2t, J=7.4, 7.8 Hz, 2H), 2.20, 2.09 (2s, 3H), 1.15-1.70(m, 4H), 0.91, 0.87 (2t, J=7.2, 7.3 Hz, 3H).

4-(N-Butyl-N-trifluoroacetylaminomethyl)-1,2-dimethoxy-naphthalene(521). Naphthalene 519 (200 mg, 0.73 mmol) was acylated withtrifluoroacetyl anhydride (210 mg, 1 mmol) in the presence of TEA (0.2mL, 1.5 mmol) by raising the temperature during 3 h from −40° to 0° C.The reaction mixture was diluted with CH₂Cl₂, washed with aqueousNaHCO₃, 3% HCl, brine and finally dried (MgSO4). The crude product (266mg, 99%) was used in the following step without further purification. ¹HNMR (CDCl₃) 8.17-8.25 (m, 1H), 7.82 (t, J=7.7 Hz, 1H), 7.40-7.55 (m,2H), 7.16, 7.03 (2s, 1H), 5.11, 5.08 (2s, 2H), 4.01, 4.03 (2s, 3H),3.98, 3.96 (2s, 3H), 3.40, 3.25 (2t, J=7.5, 7.4 Hz, 2H), 1.45-2.75 (m,2H), 1.10-1.45 (m, 2H), 0.89 (t, J=7.4, 3H).

4-[N-Butyl-N-[3-(4-morpholinocarbonyl)ethylcarbonyl]aminomethyl]-1,2-dimethoxynaphthalene(522). 3-(N-Morpholinocarbonyl)propionic acid (139 mg, 0.74 mmol) inCH₂Cl₂ (5 mL) was heated to reflux with thionyl chloride (440 mg, 3.7mmol) for 1 h and all volatiles were evaporated in vacuo. The residuewas dissolved in anhydrous CH₂Cl₂ (3 mL), cooled to 0° C. andnaphthalene 519 (100 mg, 0.37 mmol), followed by DMAP (45 mg, 0.37 mmol)and TEA (140 μL, 1 mmol) were added into the reaction mixture. Afterstirring for 1 h at room temperature the reaction was quenched with wetEtOAc (10 mL), washed with 3% HCl, aqueous NaHCO₃, brine, and dried(Na₂SO₄). Purification by chromatography (15% EtOAc in hexane) gave 522(160 mg, 98%). The product was used directly in the next step. ¹H NMR(CDCl₃) 8.19, 8.17 (2d, J=7.7, 7.8 Hz, 1H), 7.92, 7.85 (2d, J=8.2, 8.05Hz, 1H), 7.38-7.56 (m, 4H), 7.25, 7.17 (2s, 1H), 5.05, 5.03 (2s, 2H),4.03, 4.00 (2s, 3H), 3.99, 3.98 (2s, 3H), 3.25-3.82 (m, 14H), 1.15-1.82(m, 4H), 0.88. 0.85 (2t, J=7.1, 6.7 Hz, 3H).

Demethylation of dimethoxynaphthalenes with boron tribromide.4-(Butylamino=methylene)-1,4-dihydro-2-hydroxy-1-oxo-naphthalene (523).A solution of dimethoxynaphthalene 519 (30 mg, 0.11 mmol) in CH₂Cl₂ (2mL) was treated with a 1M solution of BBr₃ in CH₂Cl₂ (1.1 mL) at −78° C.and stirred at this temperature for 2 h. The reaction mixture was placedin a freezer at −10° C. for 3 h, quenched with Et₂O (1 mL) by stirringfor 15 min at room temperature and neutralized with aqueous solution ofNaHCO₃. The product was extracted with EtOAc, dried (MgSO₄), and thesolvent was removed in vacuo. The residue was dissolved in Et₂O, stirredfor 10 h in an open flask and purified by chromatography (5% MeOH inCHCl₃). Trituration with Et₂O yielded the product 523 (8 mg, 30%). ¹HNMR (CDCl₃) 9.05 (bs, 1H), 8.31 (d, J=8.1 Hz, 1H), 7.65-7.85 (m, 1H),7.05-7.65 (m, 3H), 3.20-3.60 (m, 2H), 1.50-1.85 (m, 2H), 2.25-1.50 (m,2H), 0.80-1.10 (m, 3H). HRMS (EI) 243.1250. Calcd for C₁₅H₁₇NO₂243.1259.

4-(N-Acetyl-N-butylaminomethyl)-1,2-dihydro-1,2-dioxonaphthalene (524)was prepared from dimethoxynaphthalene 520 using the procedure describedfor 523. The product (60%) was purified by chromatography (1.5% MeOH inCHCl₃) followed by trituration with Et₂O. ¹H NMR (CDCl₃) 8.1 (dd,J=7.53, 1.2 Hz, 1H), 7.67 (dd, J=7.7, 1.1 Hz, 1H), 7.50-7.62 (m, 2H),6.21 (s, 1H), 4.68 (s, 2H), 3.35 (t, J=8.0 Hz, 2H), 2.25 (s, 3H),1.50-1.75 (m, 2H), 1.15-1.50 (m, 2H), 0.96 (t, J=5.8, 3H). HRMS (EI)285.1383. Calcd for C₁₇H₁₉NO₃ 285.1365.

4-(N-Butylaminomethyl-N-trifluorocetyl)-1,2-dihydro-1,2-dioxonaphthalene(525) was obtained from dimethoxynaphthalene 521 using the proceduredescribed for 523. The product (37%) was purified by chromatography (3%MeOH in CHCl₃) followed by trituration in Et₂O. ¹H NMR (CDCl₃) 8.29 (d,J=7.13 Hz, 1H), 7.40-7.85 (m, 3H), 6.19 (s, 1H), 4.73 (s, 2H), 3.35-3.70(m, 2H), 1.50-1.80 (m, 2H), 1.35-1.80 (m, 2H), 0.96 (t, J=7.2 Hz, 3H).HRMS (EI) 339.1106. Calcd for C₁₇H₁₆F₃NO₃ 339.1082.

4-[[N-Butyl-N-(4-morpholino-4-oxobutyryl)amino]methyl]-1,2-dihydro-1,2-dioxonaphthalene(526) was obtained from dimethoxynaphthalene 522 using the proceduredescribed for 523. The product (10%) was purified by chromatography(25%-40% EtOAc in hexane) followed by trituration in Et₂O. ¹H NMR(CDCl₃) 8.19 (d, J=7.4 Hz, 1H), 7.70 (t, J=6.4 Hz, 1H), 7.59 (d, J=6.5Hz, 1H), 7.50 (t, J=7.9 Hz, 1H), 6.33 (s, 1H), 4.65 (s, 2H), 3.35-3.80(m, 14H), 1.65-1.85 (m, 2H), 1.25-1.50 (m, 2H), 0.96 (t, J=7.2 Hz, 3H).

meso-Tetra[4-[6-[(1,2-dihydro-1,2-dioxonaphth-4-yl)oxy]hexyloxy]phenyl]porphine(527). A 1 M solution of Bu₄NOH in MeOH (0.212 mL,) was added to astirred solution of meso-tetra(4-hydroxyphenyl)porphine (36 mg, 0.53mmol) in MeOH (5 mL), stirring was kept for 10 min and the mixtureconcentrated to dryness in vacuo. Naphthoquinone 505 (81 mg, 0.21 mmol)in DMF (2 mL) was added to the porphyrin, the solution stirred for 48 hand diluted with H₂O (20 mL). The product was extracted with CHCl₃,washed with brine, the solvent was evaporated and the residue wastriturated with Et₂O. Purification by flash chromatography (2-3% MeOH inCHCl₃) followed by recrystallization from CHCl₃/Et₂O (1:3) afforded theproduct as a dark red solid (19.6 mg, 21%). ¹H NMR (CDCl₃) 8.86 (s, 8H),8.01-8.15 (m, 12H), 7.9 (d, J=7.8 Hz, 4H), 7.68 (t, J=6.3 Hz, 4H), 7.55(t, J=7.5 Hz, 4H), 7.27 (d, J=7.8 Hz, 8H), 5.98 (s, 4H), 4.15-4.30 (m,16 H), 1.80-2.10 (m, 16H), 1.65-1.80 (m, 16H). Anal. Calcd forC₁₀₈H₉₄N₄O₁₆×1.5 H₂O: C, 74.87; H, 5.43; N, 3.23. Found: C, 74.62; H,5.57; N, 3.11.

meso-Tetra[4-[6-[(1,2-dihydro-1,2-dioxanaphth-4-yl)oxyhexyl]oxycarbonyl]phenyl]porphyrin(528). EDCI (518 mg, 2.7 mmol) was added at 0° C. to a mixture ofmeso-tetra(4-carboxyphenyl)porphyrin (500 mg, 0.63 mmol), alcohol 4 (831mg, 3 mmol), and DMAP (159 mg, 1.3 mmol) in CH₂Cl₂ (10 mL). The solutionwas stirred for 2 h, the cooling bath was removed and the reactionmixture was left at room temperature overnight. It was diluted withCH₂Cl₂, washed with 2% HCl, H₂O, aqueous solution of NaHCO₃, H₂O, 5%aqueous solution of NaHSO₃, H₂O, dried (Na₂SO₄) and concentrated invacuo. The analytical sample was prepared by column chromatography onsilica (2% MeOH in CHCl₃). Mp 98-110° C. (decomp.) Yield 572 mg, 50%. ¹HNMR (CDCl₃) 8.81 (s, 8H,), 8.45 (d, J=8.2 Hz, 8H), 8.30 (d, J=8.0 Hz,8H), 8.09 (d, J=6.9 Hz, 4H), 7.89 (d, J=7.3 Hz, 4H), 7.70 (t, J=7.1 Hz,4H), 7.56 (t, J=7.1 Hz, 4H), 5.98 (s, 4H), 4.56 (t, J=6.5 Hz, 8H), 4.21(t, J=6.1 Hz, 8H), 1.85-2.20 (m, 16H), 1.60-1.80 (m, 16H). MS (MALDI)1838 (M⁺+23), 1817 (M⁺+1). Anal. Calcd for C₁₁₂H₉₄N₄O₂₀×4 H₂O: C, 71.18;H, 5.40; N, 2.97. Found: C, 71.27; H, 5.24; N, 3.03.

N-Acetyl-4-(7-hydroxy-1-heptenyl)-aniline (529). A solution of 5.213 g(28.8 mmol) of 6-bromohexanol and 7.55 g (28.8 mmol) oftriphenylphosphine in 50 mL of CH₃CN was refluxed for 24 hr. Evaporationof solvent yielded the crude phosphonium salt, which was used directlyin the next reaction. The crude phosphonium salt and 4.690 g (28.7 mmol)of 4-acetamidobenzaldehyde were dissolved in a mixture of 150 mL ofCH₂Cl₂ and 150 mL of THF. To the cooled solution was added 1.529 g (60.5mmol) of 95% NaH as a slurry in CH₂Cl₂ (10 mL). The reaction mixture wasstirred in an ice bath for 1 hr, then at room temperature for 19 hr. Themixture was partitioned between 350 mL CH₂Cl₂ and 500 mL 1N HCl. Theaqueous phase was extracted with CH₂Cl₂ (4×100 mL). The CH₂Cl₂ extractswere combined, dried with MgSO₄, and evaporated to dryness. Columnchromatography on silica gel eluting first with 1% MeOH in CH₂Cl₂ andthen with 2% MeOH in CH₂Cl₂ afforded 4.913 g (69% from 6-bromohexanol)of alkene 529 as a mixture of E and Z isomers: ¹H NMR (250 MHz, CDCl₃,TMS) δ 7.5-7.4 (m, 4H), 7.3-7.1 (m, 4H), 6.4-6.3 (m, 2H), 6.2-6.1 (m,1H), 5.7-5.6 (m, 2H), 3.65 (t, J=6.5 Hz, 2H), 3.63 (t, J=6.5 Hz, 2H),2.4-2.1 (m, 4H), 2.18 (s, 3H), 2.17 (s, 3H), 1.7-1.3 (m, 12H).

4-(7-Hydroxyheptyl)-aniline (530). To a solution of 4.913 g (19.9 mmol)of N-acetyl-4-(7-hydroxy-1-heptenyl)-aniline 529 in 100 mL of 10% MeOHin CH₂Cl₂ in a Parr bottle were added 490 mg of 10% Pd/C. The bottle wasplaced on a hydrogenation apparatus and shaken for 4 hr at 25 psi ofhydrogen. Removal of catalyst by filtration through a celite pad andevaporation of solvent afforded 5.294 g of alkane: ¹H NMR (300 MHz,CDCl₃, TMS) δ 7.80 (s, NH), 7.38 (d, J=8 Hz, 2H), 7.09 (d, J=8 Hz, 2H),3.61 (t, J=6.6 Hz, 2H), 2.54 (t, J=7.6 Hz, 2H), 2.12 (s, 3H), 1.6-1.5(m, 4H), 1.4-1.3 (m, 6H). A solution of the alkane in 40 mL of MeOH wasmixed with 190 mL of 2N HCl. The reaction mixture was refluxed for 23hr. Then the reaction mixture was added to a cooled mixture of 190 mL 2NNaOH and 200 mL CH₂Cl₂. The aqueous phase was extracted with CH₂Cl₂(4×100 mL). The CH₂Cl₂ extracts were combined, dried with MgSO₄, andevaporated to dryness, to afford 3.579 g of aniline 530 (87% fromalkene): ¹H NMR (300 MHz, CDCl₃, TMS) δ 6.95 (d, J=8.3 Hz, 2H), 6.61 (d,J=8.3 Hz, 2H), 3.60 (t, J=6.6 Hz, 2H), 2.48 (t, J=7.6 Hz, 2H), 1.6-1.5(m, 4H), 1.4-1.3 (m, 6H).

N-(9-Acridinyl)-4-(7-hydroxyheptyl)-aniline (531). To a solution of636.9 mg (3.07 mmol) of 4-(7-hydroxyheptyl)-aniline 530 and 428 μL (3.07mmol) of Et₃N in 20 mL of MeOH were added 656.4 mg (3.07 mmol) of9-chloroacridine. After stirring for 7 hr at room temperature, thesolvent was evaporated. Purification by column chromatography on silicagel with 5% MeOH in CH₂Cl₂ gave 1.079 g (91%) ofN-(9-acridinyl)-4-(7-hydroxyheptyl)-aniline 531: ¹H NMR (300 MHz, CDCl₃,TMS) δ 8.0-7.9 (m, 4H), 7.63 (t, J=7 Hz, 2H), 7.3-7.2 (m, 2H), 7.07 (d,J=8.3 Hz, 2H), 6.85 (d, J=8.3 Hz, 2H), 3.64 (t, J=6.6 Hz, 2H), 2.57 (t,J=7.6 Hz, 2H), 1.7-1.5 (m, 4H), 1.4-1.3 (m, 6H).

N-(9-acridinyl)-4-(7-iodoheptyl)-aniline (532). To a solution of 604.1mg (1.57 mmol) of N-(9-acridinyl)-4-(7-hydroxyheptyl)-aniline 531 in 20mL of pyridine cooled to 0° C. was added 200 μL (2.58 mmol) ofmethanesulfonyl chloride. The reaction mixture was stirred at 0° C. for1 hr 20 min, then partitioned between 180 mL of CH₂Cl₂ and 75 mL ofwater. The aqueous phase was extracted with CH₂Cl₂ (3×30 mL). The CH₂Cl₂extracts were combined, washed with 40 mL of saturated NaCl solution,dried with MgSO₄, and evaporated to dryness.

The sulfonate was dissolved in 20 mL of acetone. To the solution wasadded 355.0 mg (2.37 mmol) of NaI, and the mixture was refluxed for 8hr, then stirred at room temperature for 16 hr. The reaction mixture waspartitioned between 200 mL of ethyl acetate and 100 mL of water. Theorganic phase was washed with 5% sodium thiosulfate (3×30 mL). Allaqueous phases were combined and backextracted with 75 mL of ethylacetate. Both ethyl acetate phases were combined, dried with MgSO₄, andevaporated to dryness, to afford 600.2 mg (77%) ofN-(9-acridinyl)-4-(7-iodoheptyl)-aniline 532: ¹H NMR (300 MHz, CDCl₃,TMS) δ 8.0-7.9 (m, 4H), 7.66 (t, J=7 Hz, 2H), 7.3-7.2 (m, 2H), 7.06 (d,J=8 Hz, 2H), 6.81 (d, J=8 Hz, 2H), 3.18 (t, J=7 Hz, 2H), 2.57 (t, J=7.6Hz, 2H), 1.9-1.8 (m, 2H), 1.7-1.7 (m, 2H), 1.4-1.3 (m, 6H).

Quinone-anilinoacridine (533) (SL-11064). To a solution of 1.554 g (3.14mmol) of N-(9-acridinyl)-4-(7-iodoheptyl)-aniline 532 in a mixture of 40mL of CHCl₃ and 2 mL of MeOH was added 1.765 g (6.28 mmol) of silversalt. The reaction mixture was refluxed for 23 hr. The reaction mixturewas diluted with CH₂Cl₂, filtered, and evaporated to dryness.Purification and separation of the para- and orthoquinone isomers wereaccomplished using a series of columns on silica gel using 5% MeOH inCH₂Cl₂, Et₂O, and 10% MeOH in CH₂Cl₂. Isolated 108.9 mg of 533 as a darkorange solid.

N-Acetyl-4-(7-methanesulfonyl-1-heptenyl)-aniline. To a cooled solutionof 500 mg (2.02 mmol) of N-acetyl-4-(7-hydroxy-1-heptenyl)-aniline 529and 0.5 mL (6.18 mmol) of pyridine in 10 mL of CH₂Cl₂ was added 240 μL(3.10 mmol) of methane-sulfonyl chloride. The reaction mixture wasstirred at room temperature for 22 hr. The reaction mixture was dilutedwith CH₂Cl₂, washed with 1N HCl (4×50 mL), washed with saturated NaClsolution (50 mL), dried with MgSO₄, and evaporated to dryness. Columnchromatography on silica gel with 5% MeOH in CH₂Cl₂ afforded 416.1 mg(63%) of mesylate (mixture of E and Z isomers): ¹H NMR (250 MHz, CDCl₃,TMS) δ 7.47 (d, J=8 Hz), 7.43 (d, J=8 Hz), 7.29 (d, J=8 Hz), 7.22 (d,J=8 Hz), 6.4-6.3 (m), 6.2-6.0 (m), 5.7-5.6 (m), 4.23 (t, J=6.6 Hz), 4.22(t, J=6.6 Hz), 2.4-2.3 (m), 2.3-2.1 (m), 2.18 (s), 2.17 (s), 1.9-1.7(m), 1.6-1.4 (m).

N-Acetyl-4-(7-iodo-1-heptenyl)-aniline (534). To a solution of 2.641 g(8.11 mmol) of N-acetyl-4-(7-methanesulfonyl-1-heptenyl)-aniline in 60mL of acetone was added 1.832 g (12.2 mmol) of NaI. The reaction mixturewas refluxed for 19 hr. Then, filtration and evaporation of solvent gave3.410 g (quant) of iodide 534, which was used as is in the nextreaction.

Phosphonium iodide (535). A solution of 3.410 g ofN-acetyl-4-(7-iodo-1-heptenyl)-aniline 534 and 2.143 g (8.17 mmol) oftriphenylphosphine in 70 mL of CH₃CN was refluxed for 43 hr. Evaporationof solvent and column chromatography on silica gel with 5% MeOH inCH₂Cl₂ yielded 4.781 g (95% from mesylate) of phosphonium iodide.

1-(3,4-Dimethoxy-1-naphthyl)-8-(4-acetamidophenyl)-1,7-octadiene (536).To a cooled solution of 3.17 g (5.12 mmol) of phosphonium iodide 535 and1.093 g (5.05 mmol) of 3,4-dimethoxy-1-naphthaldehyde 518 in 20 mL ofTHF and 25 mL of CH₂Cl₂ was added 130 mg (5.14 mmol) of 95% NaH. Thereaction mixture was stirred at room temperature for 21 hr. The mixturewas partitioned between 200 mL 1N HCl and 350 mL CH₂Cl₂. The aqueousphase was extracted with CH₂Cl₂ (6×75 mL). The CH₂Cl₂ extracts werecombined, dried with MgSO₄, and evaporated to dryness. Columnchromatography on silica gel with 1% MeOH in CH₂Cl₂ afforded 1.073 g(49%) of diene 536.

1-(3,4-Dimethoxy-1-naphthyl)-8-(4-acetamidophenyl)-octane. To a solutionof 556.3 mg (1.29 mmol) of1-(3,4-dimethoxy-1-naphthyl)-8-(4-acetamidophenyl)-1,7-octadiene 536 in20 mL of CH₂Cl₂ in a Parr bottle were added 55.4 mg of 10% Pd/C. Thebottle was placed on a hydrogenation apparatus and shaken for 2.5 hr at32 psi of hydrogen. Removal of catalyst by filtration through a celitepad and evaporation of solvent afforded 554.6 mg (99%) of octane: ¹H NMR(250 MHz, CDCl₃, TMS) δ 8.14 (d, J=8 Hz, 1H), 7.94 (d, J=8 Hz, 1H),7.5-7.4 (m, 1H), 7.4-7.3 (m, 3H), 7.12 (s 1H), 7.11 (d, J=8.2 Hz, 2H),3.99 (s, 3H), 3.98 (s, 3H), 3.0-2.9 (m, 2H), 2.6-2.5 (m, 2H), 2.16 (s,3H), 1.8-1.3 (m 12H).

1-(3,4-Dimethoxy-1-naphthyl)-8-(4-aminophenyl)-octane (537). A solutionof 554.6 mg (1.28 mmol) of1-(3,4-dimethoxy-1-naphthyl)-8-(4-acetamidophenyl)-octane in 20 mL ofMeOH was mixed with 21 mL of 2N HCl. The reaction mixture was refluxedfor 23 hr. Then the reaction mixture was partitioned between 75 mL ofCH₂Cl₂ and 21 mL of 2N NaOH. The aqueous phase was extracted with CH₂Cl₂(5×40 mL). The CH₂Cl₂ extracts were combined, dried with MgSO₄, andevaporated to dryness. Column chromatography on silica gel with 1% MeOHin CH₂Cl₂ gave 374.6 mg (75%) of aniline 537: ¹H NMR (250 MHz, CDCl₃,TMS) δ 8.14 (d, J=8 Hz, 1H), 7.94 (d, J=8 Hz, 1H), 7.47 (t, J=8 Hz, 1H),7.37 (t, J=8 Hz, 1H), 7.12 (s, 1H), 6.96 (d, J=8 Hz, 2H), 6.62 (d, J=8Hz, 2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.1-3.0 (m, 2H), 2.5-2.4 (m, 2H),1.8-1.3 (m 12H).

Naphthylacridine (538). To a solution of 99 mg (2.53×10⁻⁴ mol) of1-(3,4-dimethoxy-1-naphthyl)-8-(4-aminophenyl)-octane 537 and 35 mL(2.51×10⁻⁴ mol) of Et₃N in 4 mL of MeOH were added 54 mg (2.53×10⁻⁴ mol)of 9-chloroacridine. The reaction mixture was stirred at roomtemperature for 20 hr. Evaporation of solvent and column chromatographyon silica gel with first 1% MeOH in CH₂Cl₂ and then 3% MeOH in CH₂Cl₂afforded 118.2 mg (82%) of acridine 538: ¹H NMR (250 MHz, CDCl₃, TMS) δ8.14 (d, J=8 Hz, 1H), 8.0-7.9 (m, 5H), 7.66 (br t, 2H), 7.46 (t, J=8 Hz,1H), 7.37 (t, J=8 Hz, 1H), 7.3-7.2 (m, 2H), 7.12 (s, 1H), 7.06 (d, J=8.4Hz, 2H), 6.82 (d, J=8.4 Hz, 2H), 3.1-3.0 (m, 2H), 2.6-2.5 (m, 2H),1.8-1.3 (m, 12H).

Quinone-acridine (539) (SL-11125). To a solution of 546 mg (9.60×10⁻⁴mol) of acridine 538 in 15 mL of CH₂Cl₂ cooled to −68° C. was added 9.6mL of 1M BBr₃ in CH₂Cl₂. After 18.5 hr at −10° C., the reaction mixturewas cooled to −68° C. and 10 mL of Et₂O were added. After stirring atroom temperature for 30 min, 20 mL of saturated NaHCO₃ solution wereadded. The resulting precipitate was collected by filtration andtriturated twice with 50 mL of CH₂Cl₂ to give 555.9 mg of quinone 539:¹H NMR (250 MHz, DMSO-d₆, TMS) δ 9.11 (s), 8.59 (s), 8.14 (d, J=9 Hz),8.0-7.9 (m), 7.82 (d, J=8 Hz), 7.4-7.2 (m), 6.98 (s), 2.87 (t, J=7 Hz),2.65 (t, J=7 Hz), 1.7-1.5 (m), 1.4-1.3 (m).

N-(9-acridyl)-mesitylenesulfonamide (541). To a suspension of 4.00 g(20.6 mmol) of 9-aminoacridine 540 in 350 mL of CHCl₃ was added 2.9 mL(20.8 mmol) of Et₃N and 4.50 g (20.6 mmol) of mesitylenesulfonylchloride. The reaction mixture was refluxed for 72 hr. Then the reactionmixture was filtered and the solvent was evaporated. The material waspurified by column chromatography on silica gel by eluting first with 1%MeOH in CH₂Cl₂ and then with 5% MeOH in CH₂Cl₂ to yield 458.4 mg (6%) ofsulfonamide 541 as an orange solid: ¹H NMR (300 MHz, CDCl₃, TMS) δ 9.25(s, 1H), 8.77 (d, J=8 Hz, 2H), 7.46 (t, J=8 Hz, 2H), 7.21 (d, J=8 Hz,2H), 7.15 (t, J=8 Hz, 2H), 7.02 (s, 2H), 2.78 (s, 6H), 2.36 (s, 3H).

N-(9-acridyl)-N-(5-bromopentyl)-mesitylenesulfonamide (542). A solutionof 450 mg (1.20 mmol) of N-(9-acridyl)-mesitylenesulfonamide in 20 mL ofDMF was placed under an atmosphere of argon and cooled to 0° C. To thecooled solution was added 36 mg (1.42 mmol) of NaH (95%). The reactionmixture was stirred at 0° C for 5 min and at room temperature for 1 hr.Then the reaction mixture was cooled to 0° C., and 1.65 mL (12.1 mmol)of 1,5-dibromopentane were added. The reaction mixture was stirred at70-80° C. for 23 hr. The reaction mixture was cooled, and quenched with20 mL of water. The mixture was partitioned between CH₂Cl₂ and water.The aqueous phase was washed with CH₂Cl₂ (2×20 mL). The CH₂Cl₂ washeswere combined with the organic phase, dried with MgSO₄, and evaporatedto dryness. The material was purified by column chromatography on silicagel with CH₂Cl₂ to afford 382.2 mg (60%) of bromide 542 as an orangeoil: ¹H NMR (300 MHz, CDCl₃, TMS) δ 8.25 (d, J=9 Hz, 2H), 7.94 (d, J=9Hz, 2H), 7.76 (t, J=8 Hz, 2H), 7.45 (t, J=8 Hz, 2H), 6.87 (s, 2H),4.0-3.9 (m, 2H), 3.27 (t, J=6.5 Hz, 2H), 2.30 (s, 3H), 2.22 (s, 6H),1.8-1.6 (m, 4H), 1.4-1.3 (m, 2H).

Mesityl-acridine-quinone (543). To a solution of 632.6 mg (1.20 mmol) ofN-(9-acridyl)-N-(5-bromopentyl)-mesitylenesulfonamide 542 in 15 mL ofbenzene was added 338.4 mg (1.20 mmol) of silver salt. The reactionmixture was refluxed for 24 hr. The reaction mixture was diluted withCH₂Cl₂ and filtered to remove insoluble salts. The solvent was removedand the material was purified by column chromatography on silica gelwith Et₂O to afford 333.1 mg (45%) of ortho-quinone 543 as an orangeglassy solid: ¹H NMR (300 MHz, CDCl₃, TMS) δ 8.24 (d, J=9 Hz, 2H), 8.11(d, J=8 Hz, 1H), 7.95 (d, J=9 Hz, 2H), 7.8-7.7 (m, 3H), 7.7-7.5 (m, 2H),7.5-7.4 (m, 2H), 6.86 (s, 2H), 5.85 (s, 1H), 4.1-4.0 (m, 4H), 2.29 (s,3H), 2.21 (s, 6H), 1.9-1.5 (m, 4H), 1.5-1.4 (m, 2H).

Acridine-quinone (544) (SL-11059). Under an atmosphere of argon, 151.4mg (2.45×10⁻⁴ mol) of mesityl-acridine-quinone 543 was dissolved in 30mL of 0.1M SmI₂ in THF. Then, 2.2 mL (18.2 mmol) of DMPU were addeddropwise. The reaction mixture was refluxed for 24 hr. Filtration toremove a precipitate and evaporation of solvent yielded an orange oil,which was purified by column chromatography on silica gel with 5% MeOHin CH₂Cl₂ to afford 48.7 mg (45%) of acridine-quinone 544 as an orangeglassy solid: ¹H NMR (300 MHz, DMSO-d₆, TMS) δ 8.54 (d, J=8 Hz, 2H),7.96 (t, J=7 Hz, 2H), 7.92 (d, J=7 Hz, 1H), 7.79 (d, J=8 Hz, 2H),7.7-7.6 (m, 3H), 7.51 (t, J=8 Hz, 2H), 6.01 (s, 1H), 4.20 (t, J=6 Hz,2H), 4.13 (t, J=7 Hz, 2H), 2.1-1.9 (m, 4H), 1.7-1.6 (m, 2H).

Synthesis of Quinol Phosphates: General Procedure

To a solution of 500 mg (2.05 mmol) of 4-pentyloxy-1,2-naphthoquinone546 in 10 mL of benzene was added 2.3 mL (25.1 mmol) ofdibenzylphosphite. The reaction mixture was refluxed under nitrogen for2.5 hr, after which the benzene was removed. Column chromatography ofthe residue on silica gel with 1% MeOH in CH₂Cl₂ afforded 729.3 mg (70%)of aryldibenzylphosphate 547 (mixture of two regioisomers) as an orangeoil: R_(f)=0.51, 0.66 (1% MeOH in CH₂Cl₂); ¹H NMR (250 MHz, CDCl₃, TMS)major regioisomer δ 8.1 (d), 8.0 (br, s), 7.8 (d), 7.4 (t), 7.3-7.1 (m),6.50 (s), 5.3-5.0 (AB of ABX, δ_(A)=5.16, δ_(B)=5.08, J_(AB)=11.5 Hz,J_(AX)=8.3 Hz, J_(BX)=8.8 Hz), 4.01 (t, J=6 Hz), 2.0-1.8 (m), 1.6-1.3(m), 0.96 (t, J=7 Hz); ¹³C NMR (52 MHz, CDCl₃, TMS) both regioisomers δ153.4, 144.7, 135.6 (d, J=6.1 Hz, minor regioisomer), 134.8 (d, J=5.5Hz, major regioisomer), 128.7-127.7 (m), 127.2, 123.0, 122.2, 121.4,119.8, 99.5, 71.0 (q, J=4.8 Hz), 68.3, 28.8, 22.5.

To a solution of 1.637 g (3.23 mmol) of aryldibenzylphosphate 547 in 40mL of MeOH was added 150 mg of 10% Pd/C. The reaction mixture was placedunder an atmosphere of hydrogen (balloon) and stirred at roomtemperature for 1 hr. Removal of catalyst by filtration and evaporationof solvent afforded phosphate as a brown oil. The phosphate wasdissolved in 6 mL of benzene. Addition of 9 mL of hexane and coolinggave a precipitate. The precipitate was collected by filtration, washedwith benzene/hexane=2:3, and dried, affording 797.3 mg (76%) ofarylphosphate 548 as a gray solid; R_(f)=0.77 (MeOH); ¹H NMR (250 MHz,acetone-d₆, TMS) δ 8.13 (d, J=8 Hz, 1H), 7.96 (d, J=8 Hz, 1H), 7.49 (t,J=7 Hz, 1H), 7.32 (t, J=7 Hz, 1H), 6.59 (s, 1H), 4.13 (t, J=6 Hz, 1H),2.0-1.8 (m, 2H), 1.6-1.3 (m, 4H), 0.96 (t, J=7 Hz, 3H); ¹³C NMR (52 MHz,acetone-d₆, TMS) δ 153.3 (d, J=1.3 Hz), 145.8 (narrow t), 129.3 (d,J=3.3 Hz), 127.4, 123.2, 122.2, 121.6, 120.9, 100.0, 68.7, 29.2, 28.7,22.7, 13.9.

Ethyl 2′-acetyl-5′-methoxyphenylacetate (550) Acetyl chloride (21.3 mL,300 mmol) was added to a mixture of AlCl₃ (26.7 g, 200 mmol) and ethyl3′-methoxyphenylacetate (549, 28.66 g, 147.6 mmol) in CS₂ (200 mL) at 0°C. The ice bath was removed and the mixture was allowed to warm to 20°C. with HCl gas bubbling out. After stirring at 20° C. for 30 min, themixture was refluxed for 30 min. Upon cooling down, the mixture wasadded ice (200 g) and aqueous 2 N HCl (400 mL). The resulting mixturewas extracted with ethyl acetate (2×200 mL). The extracts were washedwith water (2×100 mL), dried over MgSO₄ and concentrated in vacuo. Theresidue was crystallized from a mixture of ethyl acetate (20 mL) andhexanes (60 mL) to afford 550 (30.60 g, 88%): ¹H NMR (CDCl₃) δ 7.84 (1H,d, J=8.6 Hz), 6.86 (1H, dd, J=8.6, 2.6 Hz), 6.75 (1H, d, J=2.6 Hz), 4.17(2H, q, J=7.1 Hz), 3.92 (2H, s), 3.86 (3H, s), 2.55 (3H, s), 1.28 (3H,t, J=7.1 Hz); ¹³C NMR (CDCl₃) δ 199.04 (s), 171.44 (s), 162.22 (s),137.70 (s), 132.97 (d), 129.48 (s), 118.68 (d), 111.84(d), 60.60(t),55.39 (q), 41.17 (t), 28.39 (q), 14.24 (q).

2-Hydroxy-7-methoxy-1,4-naphthoquinone(551). Sodium ethoxide (10.40 g,150 mmol) was added to a suspension of 550 (30.45 g, 128.90 mmol) inabsolute alcohol (200 mL) at 20° C. After stirring the mixture for 1 h,air was bubbled in for 20 h. The mixture was concentrated in vacuo. Theresidue was dissolved in water (500 mL), and extracted with diethylether (200 mL). The ether layer was counter-extracted with water (50mL). The combined aqueous phase was acidified with concentrated HCl (30mL). The mixture was filtered to afford 551 (14.42 g, 55%): ¹H NMR(DMSO-d₆) δ 11.56 (1H, s, br), 7.89 (1H, d, J=8.5 Hz), 7.42 (1H, d,J=2.8 Hz), 7.36 (1H, dd, J=8.5, 2.8 Hz), 6.10 (1H, s), 3.92 (3H, s); ¹³CNMR (DMSO-d6) δ.184.07 (s), 181.20 (s), 162.92 (s), 159.16 (s), 132.35(s), 127.82 (d), 125.16(s), 120.02 (d), 110.85 (s), 109.94(d), 55.90(q).

7-Methoxy-lapachol (552). A mixture of K₂CO₃ (30 mmol) and 551 (10.21 g,50 mmol) in HMPA (100 mL) was stirred for 30 min, when it became asuspension. Dimethylallyl bromide (8.7 mL, 75 mmol) and KI (4.15 g, 25mmol) were added, and stirring was continued for 20 h at 20° C. Themixture was diluted with ice water (600 mL) and concentrated HCl (30mL), and extracted with ethyl acetate (2×200 mL). Some solid wascollected by filtration to afford the first portion of 553 (0.628 g): ¹HNMR (CDCl₃) δ 8.01 (1H, d, J=8.6 Hz), 7.56 (1H, d, J=2.7 Hz), 7.20 (1H,dd, J=8.6, 2.7 Hz), 6.09 (1H, s), 5.49 (1H, t, J=6.8 Hz), 4.57 (2H, d,J=6.8 Hz), 3.94 (3H), s), 1.81 (3H, s), 1.76 (3H, S). The ethyl acetateextracts were pooled, extracted with saturated NaHCO₃ (2×150 mL), andthe resultant aqueous extracts were acidified with concentrated HCl andfiltered to recover 551 (2.10 g, 21%).

The main ethyl acetate extract was concentrated in vacuo. The residuewas dissolved in a mixture of 1 N NaOH (500 mL) and diethyl ether (300mL). After separation, the organic layer was extracted with 1 N NaOH(100 mL) and concentrated in vacuo. The residue was chromatographed onsilica gel (10% ethyl acetate in hexanes) to afford a second portion of553 (3.43 g, 30% total).

The NaOH extracts were acidified by concentrated HCl (50 mL), andextracted with ethyl acetate (2×200 mL). The pooled extracts were dried(MgSO₄), concentrated in vacuo, and the residue was purified bychromatography on silica gel (10% ethyl acetate in hexanes) to afford552 (4.39 g, 32%): ¹H NMR (CDCl₃) δ 8.05 (1H, d, J=8.6 Hz), 7.51 (1H, d,J=2.7 Hz), 7.20 (1H, dd, J=8.6, 2.7 Hz), 7.18 (OH, s), 5.20 (1H, tt,J=6.7, 1.5 Hz), 3.93 (3H, s), 3.29 (2H, d, J=7.2 Hz), 1.79 (3H, s), 1.68(3H, s); ¹³C NMR (CDCl₃) δ 183.99 (s), 181.85 (s), 163.28 (s), 152.51(s), 133.71 (s), 131.18 (s), 129.04 (d), 126.23 (s), 123.28 (s), 120.69(d), 119.82 (d), 109.82 (d), 55.89 (q), 25.77 (q), 22.60 (t), 17.90 (q).

8-Methoxy-β-lapachone (554) Concentrated H₂SO₄ (25 mL) was added tocompound 552 (2.454 g) at 20° C. After stirring for 20 min, the mixturewas diluted with ice water (500 mL). The resulting red precipitate 554was collected by filtration, washed with water, and dried in vacuo. Itwas obtained as a red powder (2.36 g, 96%): ¹H NMR (CDCl₃) δ 7.72 (1H,d, J=8.6 Hz), 7.56 (1H, d, J=2.7 Hz), 7.12 (1H, dd, J=8.6, 2.7 Hz), 3.90(3H, S), 2.55 (2H, t, J=6.7 Hz), 1.84 (2H, t, J=6.7 Hz), 1.46 (6H, S).

8-Hydroxy-β-lapachone (555) Boron tribromide (15.0 mL, 1.0 M in CH₂Cl₂)was added to a solution of 554 (1.05 g, mmol) in anhydrous CH₂Cl₂ (40mL) at 0° C. After stirring for 15 min, the mixture was allowed to warmto 20° C. and kept stirring for 2 h. Ice water (500 mL) was added, themixture was extracted with CHCl₃ (3×100 mL), the combined extracts weredried, and concentrated in vacuo. The residue was treated withconcentrated H₂SO₄ (20 mL) at 20° C. The mixture was diluted with icewater (500 mL) and extracted with CHCl₃ (3×100 mL). The combinedextracts were reextracted with aqueous 5% NaHSO₃ (3×150 mL). The aqueousextracts were acidified with concentrated HCl (100 mL), and extractedwith CHCl₃ (3×150 mL). The extracts were dried and concentrated toafford 555 (270 mg, 27%): ¹H NMR (CDCl₃) δ 9.81 (OH, s), 7.64 (1H, d,J=8.5 Hz), 7.49 (1H, d, J=2.6 Hz), 7.06 (1H, dd, J=8.5, 2.6 Hz), 2.51(2H, t, J=6.6 Hz), 1.84 (2H, t, J=6.6 Hz), 1.45 (6H, s); HRMS (m/z)calcd for C₁₅H₁₄O₄ 258.0892, found 258.0885.

Preparation of 1,2-Naphthoquinone Bisulfite Adducts

General Procedure I. The quinone was dissolved in 10% NaHSO₃. Afterstanding for several hours at room temperature or with cooling, thequinone-bisulfite adduct precipitated. The quinone-bisulfite wascollected by filtration and dried. The quinone-bisulfite was stablizedwith addition of 300% its weight of sodium bisulfite.

General Procedure II. The quinone is dissolved in 10% NaHSO₃ in a volumeof solution such that there is no more than 300% weight excess of NaHSO₃(relative to quinone-bisulfite). When the quinone-bisulfite did notprecipitate, it was recovered from the solution by evaporation of thewater in vacuo. This procedure gives a quinone-bisulfite adduct with a300% weight excess NaHSO₃.

Synthesis of Morpholino-Ser-Lys-Leu-Gln-β-Ala-β-Lapachone (Scheme 513)

Boc-Gln-β-Ala-β-Lapachone

To a solution of 1.000 g (2.437 mmol) of β-Ala-β-Lapachone-TFA salt(SL-11006) and 600.3 mg (2.437 mmol) of Boc-Gln in 10 mL of DMF wasadded 395.3 mg (2.925 mmol) of 1-hydroxybenzotriazole. The mixture wascooled in an ice bath. Then 270 μL (2.456 mmol) of N-methylmorpholinewere added, followed by 553.0 mg (2.680 mmol) of DCC. The reactionmixture was stirred in the ice bath for 30 min and at room temperaturefor 6.5 hr. The reaction mixture was then diluted with CH₂Cl₂ andfiltered. The filtrate was washed with saturated NaHCO₃ (50 mL), with 5%citric acid (3×50 mL), with saturated NaHCO₃ (2×50 mL), with saturatedNaCl (50 mL), dried with MgSO₄, and evaporated to dryness. Purificationby column chromatography on silica gel with 5% MeOH in CH₂Cl₂ afforded692.7 mg (51%) of peptide as an orange glassy solid: R_(f)=0.11 (5% MeOHin CH₂Cl₂); ¹H NMR (250 MHz, acetone-d₆, TMS) δ 8.00 (dd, J=7.6, 1.3 Hz,1H), 7.9-7.7 (m, 2H), 7.64 (td, J=7.6, 1.3 Hz, 1H), 7.5-7.4 (br d, NH),6.9 (br s, NH), 6.2 (br s, NH), 5.2-5.1 (m, 1H), 4.1-4.0 (m, 1H),3.5-3.4 (m, 2H), 2.7-2.5, (m, 4H), 2.3-2.2 (m, 2H), 2.0-1.8 (m, 2H),1.53 (s, 3H), 1.51 (s, 3H), 1.39 (s, 9H); ¹³C NMR (52 MHz, acetone-d₆,TMS) δ 179.8, 178.8, 175.0, 172.5, 171.6, 160.8, 156.2, 111.1, 135.6,133.0, 131.6, 131.2, 128.7, 124.8, 80.8, 80.3, 79.2, 70.2, 54.8, 35.6,34.7, 32.1, 28.4, 24.8, 23.2, 23.1.

Gln-β-Ala-β-Lapachone

To a solution of 681.9 mg (1.223 mmol) of Boc-Gln-β-Ala-β-Lapachone in10 mL of CH₂Cl₂ was added 10 mL of TFA. The reaction mixture was stirredat room temperature for 25-30 min. The solvent was removed in vacuo.Column chromatography on silica gel with 10-20% MeOH in CH₂Cl₂ afforded578.5 mg (83%) of the TFA salt as an orange glassy solid: R_(f)=0.55(BuOH/H₂O/AcOH=5:3:2), 0.05 (10% MeOH in CH₂Cl₂), 0.24 (5% MeOH inCH₂Cl₂).

Boc-Leu-Gln-β-Ala-β-Lapachone

To a solution of 650.2 mg (1.138 mmol) of Gln-β-Ala-β-Lapachone-TFA saltand 263.0 mg (1.138 mmol) of Boc-Leu in 4.6 mL of DMF was added 184.5 mg(1.365 mmol) of 1-hydroxybenzotriazole. The mixture was cooled in an icebath. Then 130 μL (1.182 mmol) of N-methylmorpholine were added,followed by 258.4 mg (1.252 mmol) of DCC. The reaction mixture wasstirred in the ice bath for 30 min and at room temperature for 6.5 hr.The reaction mixture was then diluted with CH₂Cl₂ and filtered. Thefiltrate was washed with saturated NaHCO₃ (30 mL), with 5% citric acid(4×30 mL), with saturated NaHCO₃ (3×30 mL), with saturated NaCl (30 mL),dried with MgSO₄, and evaporated to dryness. Purification by columnchromatography on silica gel with 5% MeOH in CH₂Cl₂ afforded 396.9 mg(51%) of peptide as a yellow-orange glassy solid: R_(f)=0.11 (5% MeOH inCH₂Cl₂), 0.45 (10% MeOH in CH₂Cl₂), 0.81 (20% MeOH in CH₂Cl₂), 0.78(BuOH/H₂O/AcOH=5:3:2); ¹H NMR (250 MHz, acetone-d₆, TMS) δ 8.00 (d,J=7.5 Hz, 1H), 7.9-7.7 (m, 2H), 7.64 (t, J=7.5 Hz, 1H), 7.5 (br d, NH),6.9 (br s, NH), 6.3 (br s, NH), 5.2-5.1 (m, 1H), 4.4-4.2 (m, 1H),4.1-4.0 (m, 1H), 3.6-3.3 (m, 2H), 2.7-2.5 (m, 4H), 2.3-2.2 (m, 2H),2.0-1.8 (m, 2H), 1.8-1.7 (m, 1H), 1.6-1.5 (m, 2H), 1.53 (s, 3H), 1.51(s, 3H), 1.39 (s, 9H), 1.0-0.9 (m, 6H); ¹³C NMR (52 MHz, acetone-d₆,TMS) δ 179.9, 179.0, 175.2, 173.4, 172.0, 171.5, 160.9, 156.8, 135.7,133.1, 131.6, 131.2, 128.8, 124.9, 111.2, 80.9, 80.4, 79.5, 70.3, 54.5,53.5, 41.7, 35.8, 34.8, 32.1, 28.5, 27.8, 25.4, 24.9, 23.4, 23.2, 21.9.

Leu-Gln-β-Ala-β-Lapachone

To a solution of 317.0 mg (4.725×10⁻⁴ mol) ofBoc-Leu-Gln-β-Ala-β-Lapachone in 4 mL of CH₂Cl₂ was added 4 mL of TFA.The reaction mixture was stirred at room temperature for 25-30 min. Thesolvent was removed in vacuo. Column chromatography on silica gel with20% MeOH in CH₂Cl₂ afforded 277.3 mg (86%) of the TFA salt as an orangeglassy solid: R_(f)=0.17 (10% MeOH in CH₂Cl₂), 0.39 (20% MeOH inCH₂Cl₂), 0.74 (BuOH/H₂O/AcOH=5:3:2).

Nα-Boc-Lys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone

To a solution of 277.3 mg (4.050×10⁻⁴ mol) ofLeu-Gln-β-Ala-β-Lapachone-TFA salt and 168.0 mg (4.049×10⁻⁴ mol) ofNα-Boc-Lys(Nε-Cbz) in 1.6 mL of DMF was added 65.7 mg (4.862×10⁻⁴ mol)of 1-hydroxybenzotriazole. The mixture was cooled in an ice bath. Then50 μL (4.548×10⁻⁴ mol) of N-methylmorpholine were added, followed by91.9 mg (4.454×10⁻⁴ mol) of DCC. The reaction mixture was stirred in theice bath for 30 min and at room temperature for 6.5 hr. The reactionmixture was then diluted with 2 mL of CHCl₃ and filtered. The filtratewas washed with saturated NaHCO₃ (20 mL), with 5% citric acid (4×20 mL),with saturated NaHCO₃ (3×20 mL), with saturated NaCl (2×20 mL), driedwith MgSO₄, and evaporated to dryness. Purification by columnchromatography on silica gel with 10% MeOH in CH₂Cl₂ afforded 167.5 mg(42%) of peptide as an orange glassy solid: R_(f)=0.08 (5% MeOH inCH₂Cl₂), 0.44 (10% MeOH in CH₂Cl₂); ¹H NMR (250 MHz, DMSO-d₆, TMS) δ8.0-7.7 (m, 6H, quinone-H5, H6, H7, H8, & NH's), 7.7-7.6 (m, NH),7.5-7.4 (m, 2H, Cl-Cbz), 7.4-7.3 (m, 2H, Cl-Cbz), 7.20 (br s, NH), 6.73(br s, NH), 6.90 (br d, J=7.9 Hz, NH), 5.07 (s, 3H), 4.3-4.2 (m, 1H),4.2-4.1 (m, 1H), 3.9-3.8 (m, 1H), 3.3-3.2 (m, 2H), 3.0-2.9 (m, 2H),2.8-2.7 (m, 2H), 2.6-2.4 (m, 2H), 2.1-2.0 (m, 2H), 1.8-1.3 (m, 11H),1.43 (s, 3H), 1.39 (s, 3H), 0.85 (d, J=6.5 Hz, 3H), 0.81 (d, J=6.6 Hz,3H); ¹³C NMR (52 MHz, DMSO-d₆, TMS) δ 178.6, 177.8, 173.5, 173.4, 172.0,171.7, 171.0, 170.5, 162.2, 155.7, 134.9, 134.5, 132.2, 131.4, 130.9,129.9, 129.5, 129.2, 127.9, 127.2, 123.7, 79.7, 79.3, 78.0, 68.9, 62.4,54.2, 52.0, 50.8, 40.7, 35.7, 33.5, 31.2, 30.7, 29.0, 28.1, 27.8, 24.1,23.9, 23.0, 22.8, 22.7. 22.1, 21.4.

Lys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone

To a suspension of 203.1 mg (2.099×10⁻⁴ mol) ofBoc-Lys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone in 2 mL of CHCl₃ was added 1.7mL of TFA (material dissolved). The reaction mixture was stirred at roomtemperature for 20-25 min. The solvent was removed in vacuo. Columnchromatography on silica gel with 20% MeOH in CH₂Cl₂ afforded 202.0 mg(98%) of the TFA salt as an orange glassy solid: R_(f)=0.10 (10% MeOH inCH₂Cl₂), 0.40 (20% MeOH in CH₂Cl₂).

Morpholino-Ser(OBn)-Lys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone

To a solution of 194.8 mg (1.985×10⁻⁴ mol) ofLys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone-TFA salt and 61.2 mg (1.985×10⁻⁴mol) of morpholino-Ser(OBn) in 1.0 mL of DMF was added 32.2 mg(2.383×10⁻⁴ mol) of 1-hydroxybenzotriazole. The mixture was cooled in anice bath. Then 23 μL (2.092×10⁻⁴ mol) of N-methylmorpholine were added,followed by 45.1 mg (2.186×10⁻⁴ mol) of DCC. The reaction mixture wasstirred in the ice bath for 35 min and at room temperature for 6 hr. Thereaction mixture was then diluted with 2 mL of CH₂Cl₂ and filtered. Thefiltrate was washed with 5% citric acid (3×20 mL), with saturated NaHCO₃(3×20 mL), with saturated NaCl (20 mL), dried with MgSO₄, and evaporatedto dryness. Purification by column chromatography on silica gel with 10%MeOH in CH₂Cl₂ afforded 83.3 mg (36%) of peptide as an orange glassysolid: R_(f)=0.05 (5% MeOH in CH₂Cl₂), 0.41 (10% MeOH in CH₂Cl₂); ¹H NMR(250 MHz, acetone-d₆, TMS) δ 8.0-7.7 (m, 7H, quinone-H5, H6, H7, H8,NH's), 7.7-7.6 (m, NH), 7.5-7.2 (m, 10H, Cl-Cbz, OBn, NH), 6.75 (br s,NH), 6.60 (br d, J=7.1 Hz, NH), 5.07 (s, 3H), 4.49 (s, 2H), 4.4-4.3 (m,1H), 4.3-4.0 (m, 3H), 3.7-3.6 (m, 2H), 3.6-3.5 (m, 4H), 3.3-3.2 (m, 6H),3.0-2.9 (m, 2H), 2.8-2.7 (m, 2H), 2.5-2.4 (m, 2H), 2.1-2.0 (m, 2H),1.8-1.3 (m, 11H), 1.43 (s, 3H), 1.38 (s, 3H), 0.82 (d, J=6.0 Hz, 3H),0.78 (d, J=6.1 Hz, 3H).

Morpholino-Ser-Lys-Leu-Gln-β-Ala-β-Lapachone (SL-11147)

To a solution of 78.3 mg (6.763×10⁻⁵ mol) ofmorpholino-Ser(OBn)-Lys(Nε-Cbz)-Leu-Gln-β-Ala-β-Lapachone in 1.5 mL ofMeOH/CH₂Cl₂=1:9 was added 30.6 mg 10% Pd/C. Then 0.5 mL of MeOH and onedrop of HCl were added. The reaction mixture was placed under anatmosphere of H₂ (balloon) and stirred at room temperature for 16 hr.Removal of catalyst by filtration and evaporation of solvent afforded64.5 mg of crude quinone-tetrapeptide. The material was purified by prepHPLC to yield 14.4 mg (24%): R_(f)=0.04 (20% MeOH in CH₂Cl₂).

N-Fmoc-Ser(OBn)t-butyl Ester

Isobutylene was condensed into a 500 mL pressure bottle until the volumewas between 30 and 40 mL. A solution of 3.02 g (7.23 mmol) ofN-Fmoc-Ser(OBn) in 20 mL of THF was added, followed by 2 mL ofconcentrated H₂SO₄. The bottle was securely stoppered and shaken at roomtemperature for 24 hr. The reaction mixture was poured into an ice-coldmixture of 150 mL of ethyl acetate and 150 mL of saturated NaHCO₃. Theorganic phase was washed with water (3×50 mL) and dried with MgSO₄. Thesolvent was removed, and column chromatography on silica gel with CH₂Cl₂afforded 2.453 g (72%) of t-butyl ester as a colorless oil: ¹H NMR (250MHz, acetone-d₆, TMS) δ 7.85 (d, J=7.5 Hz, 2H), 7.74 (d, J=7.3 Hz, 2H),7.5-7.3 (m, 9H), 6.71 (br d, J=8.6 Hz, NH), 4.55 (ABq, δ_(A)=4.57,δ_(B)=4.52, J_(AB)=12 Hz, 2H), 4.4-4.2 (m, 4H), 3.9-3.7 (AB of ABX,δ_(A)=3.89, δ_(B)=3.75, J_(AB)=9.5 Hz, J_(AX)=4.6 Hz, J_(BX)=3.6 Hz,2H); ¹³C NMR (52 MHz, acetone-d₆, TMS) δ 170.0, 156.8, 145.0, 144.9,142.0, 129.0, 128.4, 128.3, 128.2, 127.8, 126.1, 120.7, 81.9, 73.6,70.9, 67.3, 55.9, 47.9, 28.1.

Ser(OBn)t-butyl Ester

To a solution of 3.049 g (6.44 mmol) of N-Fmoc-Ser(OBn)t-butyl ester in50 mL of CH₂CL₂ was added 3 mL of piperidine. The reaction mixture wasstirred at room temperature for 2.3 hr. Removal of solvent and columnchromatography on silica gel with 5% MeOH in CH₂Cl₂ yielded 1.306 g(81%) of Ser(OBn)t-butyl ester as a colorless oil: R_(f)=0.12 (2% MeOHin CH₂Cl₂); ¹H NMR (250 MHz, acetone-d₆, TMS) δ 7.4-7.2 (m, 5H), 4.53(Abq, δ_(A)=4.55, δ_(B)=4.52, J_(AB)=12 Hz, 2H), 3.7-3.6 (m, AB of ABX,δ_(A)=3.68, δ_(B)=3.61, J_(AB)=12 Hz, J_(AX)=4.9 Hz, J_(BX)=4.4 Hz, 2H),3.5-3.4 (m, X of ABX, δ_(X)=3.45, 1H), 1.43 (s, 9H) ; ¹³C NMR (52 MHz,acetone-d₆, TMS) δ 173.9, 139.5, 128.9, 128.2, 128.1, 80.7, 73.8, 73.5,56.2, 28.1.

Morpholino-Ser(OBn)t-butyl Ester

To a solution of 140.6 mg (5.59×10⁻⁴ mol) of Ser(OBn)t-butyl ester in 4mL of pyridine was added 66 μL (5.66×10⁻⁴ mol) of 4-morpholinecarbonylchloride. After stirring for 1 hr, the reaction mixture was partitionedbetween 75 mL of CH₂Cl₂ and 60 mL of water. The organic phase was washedwith saturated NaHCO₃ (50 mL), with 1N HCl (2×50 mL), with saturatedNaCl (50 mL), dried with MgSO₄, and evaporated to dryness. The crudeamide was purified by column chromatography on silica gel with ethylacetate to yield 80.9 mg (40%) of amide as a light orange oil:R_(f)=0.58 (ethyl acetate), 0.60 (5% MeOH in CH₂Cl₂); ¹H NMR (250 MHz,acetone-d₆, TMS) δ 7.4-7.2 (m, 5H), 5.8 (br d, NH), 4.53 (Abq,δ_(A)=4.55, δ_(B)=4.52, J_(AB)=12 Hz, 2H), 4.5-4.4 (m, X of ABX,δ_(X)=4.47, 1H), 3.9-3.6 (m, AB of ABX, δ_(A)=3.86, δ_(B)=3.69,J_(AB)=9.4 Hz, J_(AX)=4.4 Hz, J_(BX)=3.7 Hz, 2H), 3.63-3.58 (m, 4H),3.4-3.3 (m, 4H), 1.44 (s, 9H); ¹³C NMR (52 MHz, acetone-d₆, TMS) δ170.9, 157.9, 139.2, 129.0, 128.3, 128.2, 81.5, 73.5, 71.3, 67.0, 55.5,44.9, 28.1.

Morpholino-Ser(OBn)

A solution of 80 mg (2.195×10⁻⁴ mol) of morpholino-Ser(OBn) t-butylester in a mixture of 1.5 mL of CH₂Cl₂ and 1.5 mL of TFA was stirred atroom temperature for 30 min. The solvent was removed in vacuo and theremaining TFA was removed by repeated evaporation with acetone. Theresidue was triturated with Et₂O. The material was then filtered, washedwith Et₂O, washed with 0.5 mL acetone, washed again with Et₂O, and driedto yield 41.8 mg (62%) of amino acid as an off-white solid: R=0.72(BuOH/H₂O/AcOH=5:3:2); ¹H NMR (250 MHz, acetone-d₆, TMS) δ 7.4-7.3 (m,5H), 6.0-5.9 (br d, NH), 4.6-4.5 (m, 3H, OCH₂Ph & X of ABX), 3.95-3.75(m, AB of ABX, δ_(A)=3.90, δ_(B)=3.73, J_(AB)=9.6 Hz, J_(AX)=4.9 Hz,J_(BX)=3.9 Hz, 2H), 3.6-3.5 (m, 4H), 3.4-3.3 (m, 4H); ¹³C NMR (52 MHz,DMSO-d₆, TMS) d 172.4, 157.2, 138.2, 128.2, 127.4, 127.4, 72.0, 69.5,65.9, 53.8, 43.9.

Synthesis of Morpholino-Ser-Lys-Leu-Gln-Leu-β-Lapachone (Scheme 514)

Boc-Leu-β-Lapachone

A solution of 2.820 g (12.20 mmol) of Boc-Leu and 1.976 g (12.19 mmol)of 1,1-carbonyldiimidazole in 33 mL of DMF was stirred at roomtemperature for 20 min. To the solution was added 2.100 g (8.130 mmol)of 3-hydroxy-p-lapachone followed by 1.6 mL (10.70 mmol) of DBU. Afterstirring at room temperature for 5 hr, the reaction mixture waspartitioned between 200 mL of water and 200 mL of CHCl₃. The aqueousphase was washed with CHCl₃ (4×50 mL). The CHCl₃ extracts were combined,dried with MgSO₄, and evaporated to dryness. Column chromatography onsilica gel with 2% MeOH in CH₂Cl₂ afforded 2.038 g (53%) of quinone asan orange glassy solid (and mixture of two diastereomers): R_(f)=0.45(5% MeOH in CH₂Cl₂); ¹H NMR (250 MHz, acetone-d₆, TMS) δ 8.1-8.0 (m,1H), 8.0-7.9 (m, 1H), 7.9-7.8 (m, 1H), 7.7-7.6 (m, 1H), 6.34 (br d, NH),5.2-5.1 (m, 1H), 4.2-4.1 (m, 1H), 2.9-2.8 (m, 1H), 2.7-2.5 (m, 1H),1.8-1.6 (m, 3H), 1.56 (s, 1.5H), 1.53 (s, 3H), 1.52 (s, 1.5H), 1.34 (s,4.5H), 1.33 (s, 4.5H), 0.91 (d, J=7.0 Hz, 1.5H), 0.88 (d, J=6.7 Hz,1.5H), 0.84 (d, J=6.3 Hz, 1.5H), 0.82 (d, J=6.1 Hz, 1.5H).

Leu-β-Lapachone

To a solution of 2.017 mg (4.277 mmol) of Boc-Leu-β-Lapachone in 20 mLof CH₂Cl₂ was added 20 mL of TFA. The reaction mixture was stirred atroom temperature for 30 min. The solvent was removed in vacuo. Columnchromatography on silica gel with 20% MeOH in CH₂Cl₂ afforded 2.507 g(quant.) of the TFA salt as an orange glassy solid: R_(f)=0.52 (10% MeOHin CH₂Cl₂), 0.82 (20% MeOH in CH₂Cl₂); ¹H NMR (250 MHz, DMSO-d₆, TMS) δ8.6-8.5 (br s, NH), 8.0-7.9 (m, 1H), 7.9-7.8 (m, 2H), 7.7-7.6 (m, 1H),5.3-5.2 (m, 1H), 4.1-4.0 (m, 1H), 2.8-2.5 (m, 2H), 1.8-1.5 (m, 3H), 1.52(s, 1.5H), 1.49 (s, 1.5H), 1.43 (s, 3H), 0.83 (d, J=6.0 Hz, 3H), 0.66(br t, 3H); ¹³C NMR (52 MHz, DMSO-d₆, TMS) δ 178.7, 177.8, 169.2, 169.1,160.0, 159.7, 135.1, 135.1, 131.5, 131.4, 131.1, 131.0, 129.8, 129.8,127.9, 123.9, 123.8, 109.6, 109.3, 79.4, 79.1, 71.1, 70.9, 50.6, 50.4,39.0, 24.0, 23.9, 22.9, 22.3, 22.1, 22.0, 21.8, 21.7, 21.1.

Boc-Gln-Leu-β-Lapachone

To a solution of 2.235 g (3.895 mmol) of Leu-β-Lapachone-TFA salt and959.1 mg (3.894 mmol) of Boc-Gln in 15.6 mL of DMF was added 631.4 mg(4.673 mmol) of 1-hydroxybenzotriazole. The mixture was cooled in an icebath. Then 760 μL (6.912 mmol) of N-methylmorpholine were added,followed by 883.9 mg (4.284 mmol) of DCC. The reaction mixture wasstirred in the ice bath for 30 min and at room temperature for 5.8 hr.The reaction mixture was then diluted with 8 mL of CH₂Cl₂ and filtered.The filtrate was washed with 5% citric acid (3×50 mL), with saturatedNaHCO₃ (3×50 mL), with saturated NaCl (50 mL), dried with MgSO₄, andevaporated to dryness. Purification by column chromatography on silicagel with 5% MeOH in CH₂Cl₂ afforded 1.555 g (66%) of peptide as anorange glassy solid: R_(f)=0.19 (5% MeOH in CH₂Cl₂), 0.09 (5% MeOH inCHCl₃), 0.37 (10% MeOH in CHCl₃); ¹H NMR (250 MHz, DMSO-d₆, TMS) δ 8.24(br d, J=7 Hz, NH), 8.17 (br d, J=7 Hz, NH), 8.0-7.9 (m, 1H), 7.8-7.7(m, 2H), 7.7-7.6 (m, 1H), 7.22 (br s, NH), 6.83 (br d, J=8 Hz, NH), 6.76(br s, NH), 5.1-5.0 (m, 1H), 4.3-4.1 (m, 1H), 3.9-3.8 (m, 1H), 2.8-2.6(m, 1H), 2.6-2.4 (m, 1H), 2.1-2.0 (m, 2H), 1.8-1.4 (m, 5H), 1.47 (s,1.5H), 1.43 (s, 1.5H), 1.42 (s, 1.5H), 1.40 (s, 1.5H), 1.36 (s, 9H),0.86 (d, J=6.3 Hz, 1.5H), 0.79 (d, J=6.2 Hz, 1.5H), 0.73 (br t, 3H); ¹³CNMR (52 MHz, DMSO-d₆, TMS) δ 178.7, 177.8, 177.7, 173.7, 172.0, 171.7,171.5, 159.9, 159.7, 155.1, 135.1, 135.0, 131.5, 131.4, 131.0, 130.9,129.8, 129.7, 127.9, 127.8, 123.8, 109.8, 109.6, 79.5, 79.3, 77.9, 69.6,69.4, 53.7, 53.6, 50.5, 50.4, 31.4, 28.1, 27.6, 27.4, 24.2, 24.1, 24.0,22.6, 22.5, 22.1, 21.9, 21.6, 21.2.

Gln-Leu-β-Lapachone

To a solution of 1.519 g (2.533 mmol) of Boc-Gln-Leu-β-Lapachone in 12mL of CH₂Cl₂ was added 11 mL of TFA. The reaction mixture was stirred atroom temperature for 30 min. The solvent was removed in vacuo. Columnchromatography on silica gel with 20% MeOH in CH₂Cl₂ afforded 1.976 mg(quant) of the TFA salt as an orange glassy solid; ¹H NMR (250 MHz,DMSO-d₆, TMS) δ 8.97 (br d, J=6.5 Hz, NH), 8.90 (br d, J=7.0 Hz, NH),8.30 (br s, NH), 8.0-7.9 (m, 1H), 7.9-7.8 (m, 2H), 7.7-7.6 (m, 1H), 7.45(br s, NH), 6.98 (br s, NH), 5.2-5.1 (m, 1H), 4.3-4.2 (m, 1H), 3.9-3.8(m, 1H), 2.8-2.7 (m, 1H), 2.5-2.4 (m, 1H), 2.2-2.1 (m, 2H), 2.0-1.8 (m,2H), 1.7-1.5 (m, 3H), 1.49 (s, 1.5H), 1.44 (s, 1.5H), 1.42 (s, 1.5H),1.41 (s, 1.5H), 0.87 (d, J=6.3 Hz, 1.5H), 0.81 (d, J=6.3 Hz, 1.5H), 0.75(d, J=5.8 Hz, 1.5H), 0.73 (d, J=5.8 Hz, 1.5H); ¹³C NMR (52 MHz, DMSO-d₆,TMS) δ 178.7, 177.8, 177.8, 173.5, 171.3, 171.1, 168.7, 168.7, 159.9,159.8, 135.1, 131.5, 131.4, 131.1, 131.0, 129.9, 129.8, 128.0, 123.8,109.7, 109.5, 79.5, 79.3, 69.9, 69.8, 51.7, 51.6, 50.8, 50.8, 30.3,26.8, 24.2, 24.1, 22.7, 22.5. 22.2, 22.0, 21.9, 21.6, 21.2.

Boc-Leu-Gln-Leu-β-Lapachone

To a solution of 1.949 g (max 2.533 mmol) of Gln-Leu-β-Lapachone-TFAsalt and 585.7 mg (2.533 mmol) of Boc-Leu in 10 mL of DMF was added410.6 mg (3.038 mmol) of 1-hydroxybenzotriazole. The mixture was cooledin an ice bath. Then 685 μL (6.230 mmol) of N-methylmorpholine wereadded, followed by 574.7 mg (2.785 mmol) of DCC. The reaction mixturewas stirred in the ice bath for 30 min and at room temperature for 5.5hr. The reaction mixture was then diluted with CHCl₃ and filtered. Thefiltrate was washed with 5% citric acid (5×50 mL), with saturated NaHCO₃(4×70 mL), with saturated NaCl (70 mL), dried with MgSO₄, and evaporatedto dryness. Purification by column chromatography on silica gel with 5%MeOH in CHCl₃ afforded 1.221 g (68%, from Boc-Gln-Leu-β-Lapachone) ofpeptide as an orange glassy solid: R_(f)=0.09 (5% MeOH in CHCl₃), 0.29(7% MeOH in CHCl₃); ¹H NMR (250 MHz, DMSO-d₆, TMS) δ 8.36 (br d, NH),8.30 (br d, NH), 8.0-7.9 (m, 1H), 7.9-7.7 (m, 2H), 7.7-7.6 (m, 1H), 7.19(br s, NH), 6.90 (br s, NH), 6.75 (br d, NH), 5.1-5.0 (m, 1H), 4.3-4.1(m, 2H), 4.0-3.9 (m, 1H), 2.8-2.7 (m, 1H), 2.5-2.4 (m, 1H), 2.1-2.0 (m,2H), 1.8-1.4 (m, 8H), 1.47 (s, 1.5H), 1.43 (s, 1.5H), 1.41 (s, 1.5H),1.40 (s, 1.5H), 1.37 (s, 4.5H) 1.35 (s, 4.5H), 0.9-0.8 (m, 7.5H), 0.78(d, J=6.2 Hz, 1.5H), 0.73 (d, J=5.5 Hz, 1.5H), 0.71 (d, J=5.3 Hz, 1.5H);¹³C NMR (52 MHz, DMSO-d₆, TMS) δ 178.7, 177.8, 177.7, 173.6, 173.6,172.3, 171.5, 171.4, 171.3, 159.9, 159.7, 155.2, 135.0, 131.5, 131.4,131.0, 130.9, 129.8, 129.8, 127.9, 127.9, 123.8, 109.7, 109.6, 79.5,79.3, 78.0, 69.6, 69.5, 52.8, 51.4, 50.5, 50.5, 40.7, 31.2, 28.1, 24.2,24.1, 22.9, 22.6, 22.5, 22.1, 22.0, 21.9, 21.6, 21.4, 21.2.

Leu-Gln-Leu-β-Lapachone

To a solution of 1.196 g (1.678 mmol) of Boc-Leu-Gln-Leu-β-Lapachone in8 mL of CH₂Cl₂ was added 8 mL of TFA. The reaction mixture was stirredat room temperature for 30 min. The solvent was removed in vacuo. Columnchromatography on silica gel with 20% MeOH in CHCl₃ afforded 1.430 g(quant) of the TFA salt as an orange glassy solid: R_(f)=0.04 (10% MeOHin CHCl₃), 0.10 (15% MeOH in CHCl₃), 0.19 (20% MeOH in CHCl₃); ¹H NMR(250 MHz, DMSO-d₆, TMS) δ 8.46 (br d, J=6.6 Hz, NH), 8.41 (br d, J=7.2Hz, NH), 8.0-7.9 (m, 1H), 7.9-7.8 (m, 2H), 7.7-7.6 (m, 1H), 7.26 (br s,NH), 6.77 (br s, NH), 5.1-5.0 (m, 1H), 4.3-4.1 (m, 2H), 3.5-3.4 (m, 1H),2.8-2.7 (m, 1H), 2.5-2.4 (m, 1H), 2.1-2.0 (m, 2H), 1.9-1.4 (m, 8H), 1.47(s, 1.5H), 1.43 (s, 1.5H), 1.41 (s, 1.5H), 1.40 (s, 1.5H), 0.9-0.8 (m,7.5H), 0.78 (d, J=6.1 Hz, 1.5H), 0.74 (d, J=5.9 Hz, 1.5H), 0.72 (d,J=5.5 Hz, 1.5H); ¹³C NMR (52 MHz, DMSO-d₆, TMS) δ 178.7, 177.8, 177.8,173.6, 171.6, 171.4, 171.2, 159.9, 159.8, 135.1, 131.5, 131.4, 131.1,131.0, 129.9, 129.8, 127.9, 123.9, 109.8, 109.6, 79.6, 79.3, 69.6, 69.5,51.9-51.6, 51.6, 50.5, 42.3-41.8, 31.2, 28.2, 28.0, 24.2, 24.1, 23.7,22.8, 22.7, 22.6, 22.1, 21.9, 21.8, 21.6, 21.3, 21.2.

Nα-Boc-Lys(Nε-Cl-Cbz)-Leu-Gln-Leu-β-Lapachone

To absolution of 1.400 g (max 1.643 mmol) of Leu-Gln-Leu-β-Lapachone-TFAsalt and 681.6 mg (1.643 mmol) of Nα-Boc-Lys(Nε-Cl-Cbz) in 6.6 mL of DMFwas added 266.3 mg (1.971 mmol) of 1-hydroxybenzotriazole. The mixturewas cooled in an ice bath. Then 380 μL (3.456 mmol) ofN-methylmorpholine were added, followed by 372.9 mg (1.807 mmol) of DCC.The reaction mixture was stirred in the ice bath for 30 min and at roomtemperature for 5.5 hr. The reaction mixture was then diluted with CHCl₃and filtered. The filtrate was washed with 5% citric acid (4×50 mL),with saturated NaHCO₃ (4×50 mL), with saturated NaCl (65 mL), dried withMgSO₄, and evaporated to dryness. Purification by column chromatographyon silica gel with 5% MeOH in CHCl₃ afforded 897.4 mg (54%) of peptideas an orange glassy solid: R_(f)=0.10 (5% MeOH in CHCl₃); ¹H NMR (250MHz, DMSO-d₆, TMS) δ 8.31 (br d, J=7 Hz, NH), 8.25 (br d, J=7 Hz, NH),8.0-7.9 (m, 2H (1 quinone-H+1 NH)), 7.8-7.7 (m, 3H (2 quinone-H+1 NH)),7.7-7.6 (m, 1H (quinone-H)), 7.5-7.4 (m, 2H), 7.4-7.3 (m, 3H (2Cl—Ph—H+1 NH)), 7.19 (br s, NH), 6.90 (br d, J=8 Hz, NH), 6.77 (br s,NH), 5.1-5.0 (m, 4H), 4.3-4.1 (m, 3H), 3.9-3.8 (m, 1H), 3.0-2.9 (m, 2H),2.8-2.7 (m, 1H), 2.5-2.4 (m, 1H), 2.1-2.0 (m, 2H), 1.9-1.4 (m, 14H),1.47 (s, 1.5H), 1.42 (s, 1.5H), 1.41 (s, 1.5H), 1.40 (s, 1.5H), 1.37 (s,9H), 0.9-0.8 (m, 7.5H), 0.77 (d, J=6.2 Hz, 1.5H), 0.73 (d, J=5.7 Hz,1.5H), 0.70 (d, J=5.6 Hz, 1.5H); ¹³C NMR (52 MHz, DMSO-d₆, TMS) δ 178.7,177.8, 177.7, 173.6, 171.8, 171.6, 171.4, 171.3, 159.9, 159.7, 155.7,155.3, 135.0, 134.5, 132.2, 131.5, 131.4, 131.0, 130.9, 129.8, 129.8,129.5, 129.1, 127.9, 127.8, 127.2, 123.8, 109.7, 109.6, 79.5, 79.3,78.0, 69.6, 69.5, 62.4, 54.3, 51.6, 50.7, 50.5, 50.4, 41.0, 40.1, 31.3,29.0, 28.1, 27.9, 27.7, 24.2, 24.1, 24.0, 23.9, 23.0, 22.7, 22.6, 22.5,22.1, 22.0, 21.9, 21.6, 21.5, 21.2.

Lys(Nε-Cl-Cbz)-Leu-Gln-Leu-β-Lapachone

To a solution of 1.196 g (1.678 mmol) ofBoc-Lys(Nε-Cl-Cbz)-Leu-Gln-Leu-β-Lapachone in 6 mL of CH₂Cl₂ was added 5mL of TFA. The reaction mixture was stirred at room temperature for 30min. The solvent was removed in vacuo. Column chromatography on silicagel with 15% MeOH in CHCl₃ afforded 568.9 mg (65%) of the TFA salt as anorange glassy solid: R_(f)=0.09 (10% MeOH in CHCl₃), 0.23 (15% MeOH inCHCl₃), 0.38 (20% MeOH in CHCl₃); ¹H NMR (250 MHz, DMSO-d₆, TMS) δ 8.28(br d, J=7 Hz, NH), 8.23 (br d, J=7 Hz, NH), 8.1-8.0 (m, NH), 8.0-7.9(m, 2H (1 quinone-H+1 NH)), 7.8-7.7 (m, 2H), 7.7-7.6 (m, 1H), 7.5-7.4(m, 2H), 7.4-7.3 (m, 3H (2 Cl—Ph—H+1NH)), 7.23 (br s, NH), 6.78 (br s,NH), 5.1-5.0 (m, 4H), 4.3-4.1 (m, 4H), 3.0-2.9 (m, 2H), 2.8-2.7 (m, 1H),2.5-2.4 (m, 1H), 2.1-2.0 (m, 2H), 1.9-1.4 (m, 14H), 1.47 (s, 1.5H), 1.42(s, 1.5H), 1.41 (s, 1.5H), 1.39 (s, 1.5H), 0.9-0.8 (m, 7.5H), 0.77 (d,J=6.2 Hz, 1.5H), 0.73 (d, J=5.8 Hz, 1.5H), 0.71 (d, J=5.6 Hz, 1.5H); ¹³CNMR (52 MHz, DMSO-d₆, TMS) δ 178.7, 177.8, 177.7, 173.7, 171.8, 171.6,171.4, 171.3, 159.9, 159.7, 155.7, 135.0, 134.6, 132.2, 131.5, 131.4,131.0, 130.9, 129.9, 129.8, 129.5, 129.2, 127.9, 127.8, 127.2, 123.8,109.7, 109.6, 79.5, 79.3, 69.6, 69.4, 62.4, 54.4, 51.7, 50.6, 50.5,50.4, 41.1, 31.2, 29.2, 27.6, 27.5, 24.2, 24.2, 24.1, 23.0, 22.6, 22.5,22.4, 22.0, 21.9, 21.6, 21.2.

Morpholino-Ser(OBn)-Lys(Nε-Cl-Cbz)-Leu-Gln-Leu-ε-Lapachone

To a solution of 544.9 mg (5.323×10⁻⁴ mol) ofLys(Nε-Cl-Cbz)-Leu-Gln-Leu-β-Lapachone-TFA salt and 164.2 mg 5.325×10⁻⁴mol) of morpholino-Ser(OBn) in 2.15 mL of DMF was added 86.2 mg(6.379×10⁻⁴ mol) of 1-hydroxybenzotriazole. The mixture was cooled in anice bath. Then 59 μL (5.366×10⁻⁴ mol) of N-methylmorpholine were added,followed by 120.7 mg (5.850×10⁻⁴ mol) of DCC. The reaction mixture wasstirred in the ice bath for 30 min and at room temperature for 5.5 hr.The reaction mixture was then diluted with CHCl₃ and filtered. Thefiltrate was washed with 5% citric acid (4×30 mL), with saturated NaHCO₃(4×30 mL), with saturated NaCl (30 mL), dried with MgSO₄, and evaporatedto dryness. Purification by column chromatography on silica gel with 7%MeOH in CHCl₃ afforded 515.8 mg (81%) of peptide as an orange glassysolid: R_(f)=0.17 (7% MeOH in CHCl₃), 0.36 (10% MeOH in CHCl₃); ¹H NMR(250 MHz, DMSO-d₆, TMS) δ 8.22 (br d, J=7 Hz, NH), 8.18 (br d, J=7 Hz,NH), 8.0-7.9 (m, 2H (1 quinone-H+1 NH)), 7.9-7.7 (m, 3H (2 quinone-H+1NH)), 7.7-7.6 (m, 1H), 7.5-7.4 (m, 2H), 7.4-7.2 (m, 8H (2 Cl—Ph—H+5Ph—H+1 NH)), 7.20 (br s, NH), 6.78 (br s, NH), 6.60 (br d, J=7 Hz, NH),5.1-5.0 (m, 4H), 4.50 (s, 2H), 4.4-4.3 (m, 1H), 4.3-4.1 (m, 4H), 3.7-3.6(m, 2H), 3.6-3.5 (m, 4H), 3.3-3.2 (m, 4H), 3.0-2.9 (m, 2H), 2.8-2.6 (m,1H), 2.5-2.4 (m, 1H), 2.1-2.0 (m, 2H), 1.9-1.4 (m, 14H), 1.46 (s, 1.5H),1.42 (s, 1.5 H), 1.41 (s, 1.5H), 1.39 (s, 1.5H), 0.9-0.7 (m, 9H), 0.72(d, J=5.4 Hz, 1.5H), 0.70 (d, J=5.3 Hz, 1.5H); ¹³C NMR (52 MHz, DMSO-d₆,TMS) δ 178.7, 177.8, 177.7, 173.6, 171.6, 171.5, 171.4, 171.3, 171.3,170.8, 170.8, 159.9, 159.7, 157.3, 155.7, 138.2, 135.0, 134.5, 132.2,131.5, 131.4, 131.0, 130.9, 129.9, 129.8, 129.5, 129.1, 128.1, 127.9,127.8, 127.4, 127.3, 127.2, 123.8, 109.8, 109.6, 79.5, 79.3, 71.9, 69.6,69.5, 65.8, 62.4, 54.6, 52.7, 51.7, 51.0, 50.5, 50.4, 43.9, 31.3, 31.3,29.0, 27.8, 27.7, 24.2, 24.2, 24.1, 24.0, 22.9, 22.5, 22.5, 22.0, 21.8,21.6, 21.4, 21.2.

Morpholino-Ser-Lys-Leu-Gln-Leu-β-Lapachone (SL-11154)

To a solution of 486.8 mg (4.057×10⁻⁴ mol) ofmorpholino-Ser(OBn)-Lys(Nε-Cl-Cbz)-Leu-Gln-Leu-β-Lapachone in 9 mL ofMeOH/CHCl₃=1:9 was added 180.5 mg 10% Pd/C. Then two drops of HCl wereadded. The reaction mixture was placed under an atmosphere of H₂(balloon) and stirred at room temperature for 15.5 hr. Removal ofcatalyst by filtration and evaporation of solvent afforded a light brownsolid. The material was dissolved in 12 mL of MeOH/CHCl_(3=1:9,) andstirred at room temperature for 1 hr while bubbling air through thesolution. Evaporation of solvent afforded an orange glassy solid. Columnchromatography on silica gel with 20-30% MeOH in CHCl₃ yielded 52.8 mg(14%) of material as an orange solid. The material was further purifiedby prep HPLC: R_(f)=0.06 (20% MeOH in CHCl₃).

Morpholino-Ser-Lys-Leu-Gln-β-Ala-β-Lapachone (SL-11147) (depicted below)is synthesized in an analogous manner tomorpholino-Ser-Lys-Leu-Gln-β-Ala-β-Lapachone, except that the initialcoupling of Boc-Leu to 3-hydroxy-β-lapachone is replaced with couplingof Boc-β-Ala to 3-hydroxy-β-lapachone

Example 3 In Vitro Testing of the Efficacy of Novel Polyamine AnalogConjugates Against Tumor Cell Lines

The experiments described below are designed to evaluate newlysynthesized polyamine analog conjugates described above against culturedhuman prostate carcinoma cell lines LNCaP and PC-3 for their effects oncell growth, cell-cycle regulation and polyamine regulatory responses.Analogs conjugated to a PSA-recognized polypeptide moiety are evaluatedagainst LNCaP cells, which are high in PSA expression, and PC-3 cells,which are deficient in PSA expression. Polyamine analog conjugatesuseful in the present invention demonstrate specific killing in vitro ofcells expressing PSA, but not cells not expressing PSA (or a lesserextent of killing).

Model Systems and Biochemical/Cellular Assays

On the basis of the rationale built around the unique nature ofpolyamine metabolism in the prostate gland, these experiments focusprimarily, but not exclusively, on human prostatic carcinoma, moreparticularly, on two variably differentiated cell lines capable ofgrowing in culture as well as in vivo in athymic nude mice. While PC-82and LNCaP cells are high in PSA, PC-3 cells show low PSA levels. The invivo nude mice xenograft studies are carried out with the PC-82 cellline. Growth conditions for PC-82 cells in nude mouse xenograft arewell-established. Denmeade et al. (1997). It is also noted that PC-82cell line is one of the very few human prostate cancer cell lines thatproduces PSA when grown in xenograft. The LNCaP cell line is awell-differentiated prostate carcinoma originating from a primary tumorwhich is androgen-responsive, synthesizes polyamines and expressescharacteristic prostate specific antigen. Horoszewicz et al. (1983)Cancer Res. 43:1809-1818. The PC-3 carcinoma originated from a bonemetastasis and is poorly differentiated and prone to metastasis. Kaighnet al. (1979) Invest. Urol. 17:16-23. Culturing and treatment ofprostatic carcinoma cell lines, cell cycle and apoptosis determinationsbased on flow cytometry; enzyme assays including ODC, SAMDC and SSATactivities; and high pressure liquid chromatography detection andquantitation of natural polyamines and polyamine analogs are describedin the art, for example, Mi et al. (1998) Prostate 34:51-60; Kramer etal. (1997) Cancer Res. 57:5521-27; and Kramer et al. (1995) J. Biol.Chem. 270:2124-2132.

General Strategy for Analog Evaluation

Polyamine analogs are evaluated in human prostate carcinoma cellcultures for their effects on cell growth and polyamine-relatedmetabolism. Analysis begins with IC₅₀ determinations based ondose-response curves ranging from 0.1 to 1000 μM performed at 72 hr.From these studies, conditions are defined which produce about 50%growth inhibition and used to: (a) follow time-dependence of growthinhibition for up to 6 days, with particular attention to decreases incell number, which may indicate drug-induced cell death; (b)characterize analog effects on cell cycle progression and apoptosisusing flow cytometry (analysis to be performed on attached and detachedcells); (c) examine analog effects on polyamine metabolic parameters,including the biosynthetic enzymes ODC, SAMDC, the catabolic enzyme SSATand polyamine pools themselves. Analog effects are normalized tointracellular concentrations (by HPLC analysis), which also provide anindication of their relative ability to penetrate cells. Markeddifferences in analog uptake are further characterized by studyinganalog ability to utilize and regulate the polyamine transporter, asassessed by competition studies using radiolabeled spermidine, aspreviously described in Mi et al. (1998).

As shown in Table 2 and FIGS. 1-32, several novel conformationallyrestricted polyamine analogs were tested for anti-proliferativeproperties against cancer cells. Table 2 illustrates the concentrationin μM of the various novel polyamine analogs needed for 50% growthinhibition (ID₅₀) values for human cancer cell lines LNCaP, PC-3, DuPro(all three human prostate cancer cell lines), HT-29 (colon cancer cellline), A549 (lung cancer cell line), MCF7 (breast cancer cell line), andU251 MG-NCI (brain cancer cell line). FIGS. 1-32 show a representativeplot of the effects of some of these novel analogs on the growth ofhuman tumor cell lines, as determined by MTT (methyl thiazoltetrazolium) assay; known anti-proliferative polyamine analogs BE-333,BE-343, BE-444, and BE-4444 were used for, comparative purposes.

Cell Lines and Media

Human breast cancer cell line MCF7 was grown in Richter's ImprovedModified Eagle's Medium supplemented with 10% fetal bovine serum (FBS)and 2.2 g/L sodium bicarbonate. Human brain tumor cell line U251 MG-NCIwas grown in Dulbecco's Modified Eagle's Medium supplemented with 10%FBS. Human lung cancer cell line A549 was grown in Ham's F-12K medium(Cellgro, Mediatech, Inc., VA), supplemented with 10% FBS and 2 mML-glutamine. Human colon cancer cell line HT29 was cultured in McCoy's5A medium (Gibco, BRL, Gaithersburg, Md.) supplemented with 10% FBS.Human prostate cancer cell lines PC-3, LNCAP and DuPro were grown inRPMI 1640 Medium (Cellgro, Mediatech, Inc., VA) supplemented with 10%FBS. Another human prostate cancer cell line DU145 was grown inDulbecco's Modified Eagle's Medium (Gibco, BRL, Gaithersburg, Md.)supplemented with 5% FBS. The A549, MCF7, PC3, LNCAP and DuPro celllines were cultured in 100 units/mL penicillin and 100 μg/mLstreptomycin. HT29 and U251MG cell lines were grown in 50 μg/mLgentamycin (Gibco, BRL, Gaithersburg, Md.). DU145 cell line wasmaintained in 1% antibitic-antimycotic solution (Sigma, St. Louis, Mo.).The cell cultures were maintained at 37° C. in 5% CO₂/95% humidifiedair. DuPro cells were obtained from M. Eileen Dolan, University ofChicago. All other cells are available from the American Type CultureCollection, Rockville, Md.

MTT Assay

A conventional MTT assay was used to evaluate percent cell survival.Exponentially growing monolayer cells were plated in 96-well plates at adensity of 500 cells per well and allowed to grow for 24 hours. Serialdilutions of the drugs were added to the wells. Six days after drugtreatment, 25 μl of MTT solution (5 mg/ml) was added to each well andincubated for 4 hours at 37° C. Then 100 μl of lysis buffer (20% sodiumdodecyl sulfate, 50% DMF, and 0.8% acetic acid, pH 4.7) was added toeach well and incubated for an additional 22 hours. A microplate reader(“EMAX”-brand, Molecular Devices, Sunnyvale, Calif.) set at 570 nm wasused to determine the optical density of the cultures. Results areexpressed as a ratio of the optical density in drug-treated wells to theoptical density in wells treated with vehicle only.

As shown in Table 2, several polyamine analogs were tested foranti-proliferative properties against prostate cancer cells. Table 2illustrates the concentration of the various novel polyamine analogsneeded for 50% growth inhibition (ID₅₀) values for human prostate cancercell lines PC-3, DU-145 and DuPro, and other tumor cell lines. FIGS.1-32 show representative plots of the effects of some of these novelanalogs on the growth of human prostate tumor cell lines. Additionaldata on polyamines useful in the invention is provided in Reddy et al.(1998) J. Med. Chem. 41:4723-32. TABLE 2 PC-3 DU-145 DUPRO HT-29 A549MCF7 U251MG BE-4444 0.54 0.07 0.2 0.8 0.4 >31.25 NT SL-11029 24.5 0.32NT >31.25 >31.25 >31.25 >31.25 SL-11090 >31.25 >31.25NT >31.25 >31.25 >31.25 >31.25 SL-11091 >31.25 1.33NT >31.25 >31.25 >31.25 >31.25 SL-11092 >31.25 1.7NT >31.25 >31.25 >31.25 >31.25 SL-11093 14.3 0.01 0.06 0.40 0.26 0.66 NTSL-11094 >31.25 12.6 NT 28.8 >31.25 >31.25 >31.25 SL-11098 1.4 0.0180.08 0.40 0.51 >31.25 0.10 SL-11099 2.5 0.014 0.08 1.00 0.65 26.3 0.11SL-11100 4.7 0.021 0.29 2.00 2.20 >31.25 0.22 SL-11101 7.7 0.218 0.855.20 0.15 >31.25 1.70 SL-11102 >31.25 0.027 0.15 0.73 12.40 >31.25 0.15SL-11103 >31.25 2.8 NT 29.4 >31.25 >31.25 9.50 SL-11104 >31.25 9.4 NT25.8 0.43 >31.25 14.71 SL-11105 >31.25 1.6 >31.25 25.2 >31.25 >31.2525.9 SL-11108 2.2 0.13 0.98 2.00 >31.25 >31.25 2.00 SL-11114 0.70 0.1350.64 3.6 >31.25 NT NT SL-11118 1.65 0.05 0.25 0.98 0.21 NT NTSL-11119 >31.25 0.08 0.44 0.97 NT NT NT SL-11121 0.52 0.08 0.400.80 >31.25 17.0 NT SL-11122 >31.25 0.80 0.56 0.80 >31.25 >31.25 NTSL-11123 >31.25 0.51 >31.25 10.42 >31.25 >31.25 NTSL-11124 >31.25 >31.25 >31.25 >31.25 >31.25 >31.25 NT SL-11126 0.20 0.511.10 1.50 >31.25 0.70 NT SL-11127 >31.25 0.22 1.3 2.91 NT NT NT SL-111280.50 0.14 1.25 1.35 NT NT NT SL-11129 1.70 0.32 NT NT NT NT NTSL-11130 >31.25 0.43 NT NT NT NT NTNT indicates not tested.

Most of the tested compounds inhibited growth of at least one prostaticcancer cell line. From these data, we concluded that bis-ethylatedpolyamine analogs up to a certain degree of rigidity in the aliphaticbackbone can exhibit marked cytotoxicity in several prostate tumor celllines in culture.

As shown in FIGS. 57-59, polyamine alcohol SL-11141 and itscorresponding peptide conjugate SL-11155 (see Table 1 for the structuresof these two compounds) display effectiveness against tumor cell linesin vitro. This illustrates the ability of the peptide conjugates tofunction as effective prodrugs.

Example 4 Cell Culture and Drug Testing Protocol for Quinones

Cell Culture: The human lung adenocarcinoma cell line, A549, and humanprostatic cancer cell line, DUPRO, were a gift from Dr. M. Eileen Dolan,University of Chicago, Department of Medicine. A549 was grown in Ham'sF-12K medium (Fisher Scientific, Itasca, Ill.) supplemented with 10%fetal bovine serum and 2 mM L-glutamine. DUPRO was grown in RPMI-1640supplemented with 10% fetal bovine serum. The human colon carcinoma cellline, HT29, and the human breast carcinoma cell line, MCF7, wereobtained from the American Type Culture Collection, Rockville, Md. HT29cells were grown in McCoy's 5A medium (Gibco, BRL, Gaithersburg, Md.)supplemented with 10% fetal bovine serum. MCF7 cells were grown inRichter's Improved Modified Eagle's medium supplemented with 10% fetalbovine serum and 2.2 g/L sodium bicarbonate. The human prostateadenocarcinoma cell lines, LNCAP, PC-3 and DU145, were gifts from Dr.George Wilding, University of Wisconsin Comprehensive Cancer Center andthe Department of Medicine, and were grown in Dulbecco's ModifiedEagle's medium supplemented with a 5% fetal bovine serum. The malignantglioma cell line, U251MG NCI was obtained from the brain tumor tissuebank at the University of California, San Francisco Department ofNeurosurgery, and was grown in Dulbecco's Modified Eagle's mediumsupplemented wth 10% fetal bovine serum. DUPRO, A549 and MCF7 cells weregrown in 100 units/mL penicillin and 100 μg/mL streptomycin. HT29 andU251MG NCI cells were grown in 50 μg/mL gentamycin. LNCAP, PC-3 andDU145 cells were maintained in 1% antibiotic antimycotic solution(Sigma, St. Louis, Mo.). All cell cultures were maintained at 37° C. in5% CO₂/95% humidified air.

MTT assay. Exponentially growing monolayer cells were plated in 96 wellplates at a density of 500 cells/well and allowed to grow for 24 h.Serially diluted drug solutions were added such that the final drugconcentrations in the treatment media were between 0 and 35 μM. Cellswere incubated with drug at either 4 hr or 72 hr. After 4 hr and 72 hrtreatment, drugs were removed, fresh media (without) drug (100 uL) wasadded and cells were incubated for 6 days. After six days, 25 μL of aDulbecco's phosphate-buffered saline solution containing 5 mg/mL of MTT(Thiazolyl blue) (Sigma) was added to each well and incubated for 4 h at37° C. Then 100 μL of lysis buffer (20% sodium dodecyl sulfate, 50%N,N-dimethylformamide and 0.8% acetic acid, pH 4.7) was added to eachwell and incubated for an additional 22 h. A microplate reader (E max,Molecular Devices, Sunnyvale, Calif.) set at 570 nm was used todetermine the optical density. Results were plotted as a ratio of theoptical density in drug treated wells to the optical density in wellstreated with vehicle alone. Plotting and estimation of ID₅₀ values wereaccomplished with manufacturer supplied software. The data is presentedbelow in Tables 3, 4, 5 and 6. TABLE 3 ID₅₀ (μM) Values of Quinones inVarious Cultured Human Prostate Tumor Cell Lines Determined by the MTTAssay ID₅₀ (μM) of different prostate cells Quinones Structures ofQuinones PC-3 DUPRO DU145 LNCAP SL-11051

17.11 19.3 11.16 SL-11059

4.3 SL-11062

1.71 SL-11064

0.7 2.2 0.13 SL-11065

1.4 SL-11066

>31.25 SL-11067

0.25 SL-11068

1.5 SL-11074

4.6 SL-11075

2.0 SL-11076

1.8 SL-11078

18.4 SL-11079

22.5 SL-11080

7.3 SL-11081

5.6 SL-11082

5.4 SL-11083

5.2 SL-11084

5.9 SL-11085

>31.25 SL-11087

2.4 SL-11088

>31.25 SL-11089

11.03 SL-11095

4.2 SL-11096

3.6 SL-11106

>31.25 SL-11107

4.3 >31.25 17.2 SL-11112

>31.25 27.9 >31. 22.9 SL-11113

27.9 >31.25 29.2 SL-11120

6.4 13.1 3.8 SL-11125

5.9 7.9 0.13 SL-11145

1.97(4 hr) 0.51(6 days) 0.7(4 hr) 0.8(6 days) SL-11147

6.3(4 hr) 1.24(72 hr) 28.08 (4 hr) 2.01 (72 hr) SL-11148

6.3 1.84

TABLE 4 ID₅₀ (μM) Values of Quinones in Various Cultured Human TumorCell Lines Determined by the MTT Assay ID₅₀ (μM) of different Tumorcells Lung Colon Breast Brain Quinones Structures of Quinones A549 HT-29MCF7 U251-MG SL-11051

17.23 20.02 SL-11052

26.88 SL-11053

7.39 2.8 SL-11054

>31.25 >31.25 SL-11056

>31.25 >31.25 >31.25 >31.25 SL-11059

15.0 10.12 SL-11060

>31.25 >31.25 17.23 >31.25 SL-11062

18.64 SL-11064

9.3 SL-11065

2.13 SL-11066

>31.25 SL-11067

>31.25 0.53 SL-11068

24.0 SL-11074

SL-11075

SL-11076

1.8 1.7 10.24 SL-11078

18.9 19.3 30.85 SL-11079

SL-11080

SL-11081

SL-11082

SL-11083

SL-11084

SL-11085

SL-11087

19.8 6.05 4.0 SL-11088

>31.25 >31.25 >31.25 SL-11089

>31.25 SL-11095

>31.25 22.1 20.6 SL-11096

17.4 3.4 3.8 SL-11106

>31.25 SL-11107

>31.25 SL-11112

SL-11113

SL-11120

26.7 20.9 4.1 SL-11125

27.97 5.7 5.1 SL-11145

2.4(4hr) 1.0(6 days) SL-11147

SL-11148

TABLE 5 ID₅₀ (μM) Value(s) of Non-Quinone Structure in A Cultured HumanProstate Tumor Cell Line Determined by the MTT Assay ID₅₀ (μM) ofdifferent prostate cells Designation Structures of Compound PC-3 DUPRODU145 LNCAP SL-11063

>31.25

TABLE 6 ID₅₀ (μM) Values of Selected Non-Quinone Compounds in VariousCultured Humans Tumor Cell Lines Determined by the MTT Assay ID₅₀ (μM)of different Tumor Cells Lung Colon Breast Brain Designation Structuresof Non-Quinone Compounds A549 HT-29 MCF7 U251-MG SL-11055

>31.25 >31.25 >31.25 >31.25 SL-11058

>31.25 SL-11063

>31.25

Example 5 In Vivo Testing of Anti-Tumor Activity of Polyamine AnalogConjugates

Strategy and Interpretation of Data for Polyamine Analog-PSA PeptideConjugates

In evaluating polyamine analog conjugates, their in vitroanti-proliferative activity against LNCAP cells, which express PSA,relative to PC-3, which do not, is determined. The cell line PC-82expresses even higher PSA levels, but does not grow in vitro and istherefore used only for in vivo analysis. Those conjugates demonstratingdifferential anti-proliferative activity (based on IC₅₀ determinations)toward LNCaP cells, can be chosen for further development.

Analog conjugates found to have potent or mechanism-basedanti-proliferative activity in vitro towards cultured prostaticcarcinoma cells are evaluated in in vivo model systems, namely LNCaP andPC-82 prostate carcinoma xenografts, both of which express PSA. Becausethe conjugate could be rapidly cleared from the circulation, it may benecessary to intensify the treatment schedule to two or three timesdaily. In addition to assessing anti-tumor activity, as described above,free analog levels in tumor and normal tissues are determined. In theevent that meaningful anti-tumor activity is observed, these sameconjugates are used to treat PC-3 prostate carcinoma xenografts, whichdo not express PSA. These experiments are designed to confirm thespecificity of drug action, since it is expected that the activityagainst such tumors which do not express PSA, such as PC-3, will bemarkedly diminished. As above, these studies are augmented bydeterminations of free analog levels in tumor.

The first goal is to determine the relative toxicity of the analogs innon-tumor-bearing DBA/2 mice. Groups of three animals each are injectedintraperitoneally with increasing concentrations of an analog, beginningat 10 mg/kg. Toxicity as indicated by morbidity is closely monitoredover the first 24 hr. The polyamine analog, BE-333 is used as aninternal standard in these studies, since a data base has already beenestablished regarding acute toxicity via a single dose treatmentrelative to chronic toxicity via a daily×5 d schedule. Thus, in the caseof new analogs, single dose toxicity relative to BE-333 is used toproject the range of doses to be used on a daily×5 d schedule.

After the highest tolerated dosage on a daily×5 d schedule is deduced,antitumor activity is determined. Typically, PC-3 tumors aresubcutaneously implanted into nude athymic mice by trocar and allowed toreach 100-200 mm³ before initiating treatment by intraperitonealinjection daily×5 d. The LNCaP tumor requires suspension in Matrigel(Microbiological Assoc.) prior to implantation, after which it grows atapproximately a 4-day doubling time. Most conjugates are given in arange between 10 and 200 mg/kg. Conjugates are evaluated at threetreatment dosages with 10-15 animals per group (a minimum of three fromeach are used for pharmacodynamic studies, described below). Mice aremonitored and weighed twice weekly to determine tumor size and toxicity.Tumor size is determined by multi-directional measurement from whichvolume in mm³ is calculated. Tumors are followed until median tumorvolume of each group reaches 1500 mm³ (i.e., 20% of body weight), atwhich time the animals are sacrificed. Although the initial anti-tumorstudies focuses on a daily×5 d schedule, constant infusion can beperformed via Alzet pump delivery for 5 days since this scheduledramatically improves the anti-tumor activity of BE-333 against A549human large cell hung carcinoma. Sharma et al. (1997) Clin. Cancer Res.3:1239-1244.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

1-37. (canceled)
 38. A compound selected from the group consisting of

and all salts thereof.
 39. A compound according to claim 38 of theformula:

and all salts thereof.
 40. A compound according to claim 38 of theformula:

and all salts thereof.
 41. A compound according to claim 38 of theformula:

and all salts thereof.
 42. A composition of matter comprising thecompound of claim 39 and a pharmaceutically acceptable excipient.
 43. Acomposition of matter comprising the compound of claim 40 and apharmaceutically acceptable excipient.
 44. A composition of mattercomprising the compound of claim 41 and a pharmaceutically acceptableexcipient.
 45. A method of treating a disease, wherein said disease isan aberrant growth of cells characterized by a significant loss of cellproliferation control, comprising administering to an individual one ormore of the compounds of claim
 38. 46. A method of treating a disease,wherein said disease is an aberrant growth of cells characterized by asignificant loss of cell proliferation control, comprising administeringto an individual the compound of claim
 39. 47. A method of treating adisease, wherein said disease is an aberrant growth of cellscharacterized by a significant loss of cell proliferation control,comprising administering to an individual the compound of claim
 40. 48.A method of treating a disease, wherein said disease is an aberrantgrowth of cells characterized by a significant loss of cellproliferation control, comprising administering to an individual thecompound of claim
 41. 49. The method of claim 45, wherein the disease iscancer.
 50. The method of claim 49, wherein the cancer is prostatecancer.
 51. The method of claim 49, wherein the cancer is breast cancer.